Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Modelling Cryptosporidium infection in human small intestinal and lung organoids

Abstract

Stem-cell-derived organoids recapitulate in vivo physiology of their original tissues, representing valuable systems to model medical disorders such as infectious diseases. Cryptosporidium, a protozoan parasite, is a leading cause of diarrhoea and a major cause of child mortality worldwide. Drug development requires detailed knowledge of the pathophysiology of Cryptosporidium, but experimental approaches have been hindered by the lack of an optimal in vitro culture system. Here, we show that Cryptosporidium can infect epithelial organoids derived from human small intestine and lung. The parasite propagates within the organoids and completes its complex life cycle. Temporal analysis of the Cryptosporidium transcriptome during organoid infection reveals dynamic regulation of transcripts related to its life cycle. Our study presents organoids as a physiologically relevant in vitro model system to study Cryptosporidium infection.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Development of asexual and sexual stages of Cryptosporidium parvum in human small intestinal organoids.
Fig. 2: C. parvum completes its life cycle inside organoids.
Fig. 3: In vitro culture of C. parvum in human lung organoids.
Fig. 4: Transcriptome analysis of host epithelia and C. parvum.

References

  1. 1.

    Clevers, H. Modeling development and disease with organoids. Cell 165, 1586–1597 (2016).

    CAS  Article  Google Scholar 

  2. 2.

    Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141, 1762–1772 (2011).

    CAS  Article  Google Scholar 

  3. 3.

    Bouzid, M., Hunter, P. R., Chalmers, R. M. & Tyler, K. M. Cryptosporidium pathogenicity and virulence. Clin. Microbiol. Rev. 26, 115–134 (2013).

    CAS  Article  Google Scholar 

  4. 4.

    Thompson, R. C. et al. Cryptosporidium and cryptosporidiosis. Adv. Parasitol. 59, 77–158 (2005).

    CAS  Article  Google Scholar 

  5. 5.

    Current, W. L. & Garcia, L. S. Cryptosporidiosis. Clin. Lab. Med. 11, 873–897 (1991).

    CAS  Article  Google Scholar 

  6. 6.

    Hunter, P. R. & Nichols, G. Epidemiology and clinical features of Cryptosporidium infection in immunocompromised patients. Clin. Microbiol. Rev. 15, 145–154 (2002).

    Article  Google Scholar 

  7. 7.

    Checkley, W. et al. A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for Cryptosporidium. Lancet Infect. Dis. 15, 85–94 (2015).

    Article  Google Scholar 

  8. 8.

    Liu, L. et al. Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000. Lancet 379, 2151–2161 (2012).

    Article  Google Scholar 

  9. 9.

    Sponseller, J. K., Griffiths, J. K. & Tzipori, S. The evolution of respiratory cryptosporidiosis: evidence for transmission by inhalation. Clin. Microbiol. Rev. 27, 575–586 (2014).

    CAS  Article  Google Scholar 

  10. 10.

    Mor, S. M. et al. Expectoration of Cryptosporidium parasites in sputum of human immunodeficiency virus-positive and -negative adults. Am. J. Trop. Med. Hyg. 98, 1086–1090 (2018).

    Article  Google Scholar 

  11. 11.

    Kotloff, K. L. et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet 382, 209–222 (2013).

    Article  Google Scholar 

  12. 12.

    Amadi, B. et al. Effect of nitazoxanide on morbidity and mortality in Zambian children with cryptosporidiosis: a randomised controlled trial. Lancet 360, 1375–1380 (2002).

    Article  Google Scholar 

  13. 13.

    Amadi, B. et al. Reduced production of sulfated glycosaminoglycans occurs in Zambian children with kwashiorkor but not marasmus. Am. J. Clin. Nutr. 89, 592–600 (2009).

    CAS  Article  Google Scholar 

  14. 14.

    Striepen, B. Parasitic infections: time to tackle cryptosporidiosis. Nature 503, 189–191 (2013).

    Article  Google Scholar 

  15. 15.

    Lendner, M. & Daugschies, A. Cryptosporidium infections: molecular advances. Parasitology 141, 1511–1532 (2014).

    Article  Google Scholar 

  16. 16.

    Karanis, P. & Aldeyarbi, H. M. Evolution of Cryptosporidium in vitro culture. Int. J. Parasitol. 41, 1231–1242 (2011).

    CAS  Article  Google Scholar 

  17. 17.

    Karanis, P. The truth about in vitro culture of Cryptosporidium species. Parasitology https://doi.org/10.1017/S0031182017001937 (2017).

  18. 18.

    Varughese, E. A., Bennett-Stamper, C. L., Wymer, L. J. & Yadav, J. S. A new in vitro model using small intestinal epithelial cells to enhance infection of Cryptosporidium parvum. J. Microbiol. Methods 106, 47–54 (2014).

    CAS  Article  Google Scholar 

  19. 19.

    Castellanos-Gonzalez, A., Cabada, M. M., Nichols, J., Gomez, G. & White, A. C. Jr. Human primary intestinal epithelial cells as an improved in vitro model for Cryptosporidium parvum infection. Infect. Immun. 81, 1996–2001 (2013).

    CAS  Article  Google Scholar 

  20. 20.

    Morada, M. et al. Continuous culture of Cryptosporidium parvum using hollow fiber technology. Int. J. Parasitol. 46, 21–29 (2016).

    CAS  Article  Google Scholar 

  21. 21.

    DeCicco RePass, M. A. et al. Novel bioengineered three-dimensional human intestinal model for long-term infection of Cryptosporidium parvum. Infect. Immun. 85, e00731-16 (2017).

    Article  Google Scholar 

  22. 22.

    Dutta, D., Heo, I. & Clevers, H. Disease modeling in stem cell-derived 3D organoid systems. Trends Mol. Med. 23, 393–410 (2017).

    CAS  Article  Google Scholar 

  23. 23.

    McCracken, K. W. et al. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature 516, 400–404 (2014).

    CAS  Article  Google Scholar 

  24. 24.

    Ettayebi, K. et al. Replication of human noroviruses in stem cell-derived human enteroids. Science 353, 1387–1393 (2016).

    Article  Google Scholar 

  25. 25.

    Umemiya, R., Fukuda, M., Fujisaki, K. & Matsui, T. Electron microscopic observation of the invasion process of Cryptosporidium parvum in severe combined immunodeficiency mice. J. Parasitol. 91, 1034–1039 (2005).

    Article  Google Scholar 

  26. 26.

    Aldeyarbi, H. M. & Karanis, P. The fine structure of sexual stage development and sporogony of Cryptosporidium parvum in cell-free culture. Parasitology 143, 749–761 (2016).

    CAS  Article  Google Scholar 

  27. 27.

    Aldeyarbi, H. M. & Karanis, P. Electron microscopic observation of the early stages of Cryptosporidium parvum asexual multiplication and development in in vitro axenic culture. Eur. J. Protistol. 52, 36–44 (2016).

    Article  Google Scholar 

  28. 28.

    Aldeyarbi, H. M. & Karanis, P. The ultra-structural similarities between Cryptosporidium parvum and the gregarines. J. Eukaryot. Microbiol. 63, 79–85 (2016).

    Article  Google Scholar 

  29. 29.

    Fayer, R. & Xiao, L. (eds) Cryptosporidium and Cryptosporidiosis 2nd edn (CRC Press, Boca Raton, 2008).

  30. 30.

    Ernest, J. A., Blagburn, B. L., Lindsay, D. S. & Current, W. L. Infection dynamics of Cryptosporidium parvum (Apicomplexa: Cryptosporiidae) in neonatal mice (Mus musculus). J. Parasitol. 72, 796–798 (1986).

    CAS  Article  Google Scholar 

  31. 31.

    Shahiduzzaman, M., Dyachenko, V., Obwaller, A., Unglaube, S. & Daugschies, A. Combination of cell culture and quantitative PCR for screening of drugs against Cryptosporidium parvum. Vet. Parasitol. 162, 271–277 (2009).

    CAS  Article  Google Scholar 

  32. 32.

    Riggs, M. W. & Perryman, L. E. Infectivity and neutralization of Cryptosporidium parvum sporozoites. Infect. Immun. 55, 2081–2087 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Sachs, N. et al. Long-term expanding human airway organoids for disease modelling. Preprint at bioRxiv https://doi.org/10.1101/318444 (2018).

  34. 34.

    Beiting, D. P. Protozoan parasites and type I interferons: a cold case reopened. Trends Parasitol. 30, 491–498 (2014).

    CAS  Article  Google Scholar 

  35. 35.

    Barakat, F. M., McDonald, V., Foster, G. R., Tovey, M. G. & Korbel, D. S. Cryptosporidium parvum infection rapidly induces a protective innate immune response involving type I interferon. J. Infect. Dis. 200, 1548–1555 (2009).

    CAS  Article  Google Scholar 

  36. 36.

    Frenal, K. & Soldati-Favre, D. Role of the parasite and host cytoskeleton in Apicomplexa parasitism. Cell Host Microbe 5, 602–611 (2009).

    CAS  Article  Google Scholar 

  37. 37.

    Elliott, D. A. et al. Cryptosporidium parvum infection requires host cell actin polymerization. Infect. Immun. 69, 5940–5942 (2001).

    CAS  Article  Google Scholar 

  38. 38.

    Rayamajhi, M., Humann, J., Penheiter, K., Andreasen, K. & Lenz, L. L. Induction of IFN-αβ enables Listeria monocytogenes to suppress macrophage activation by IFN-γ. J. Exp. Med. 207, 327–337 (2010).

    CAS  Article  Google Scholar 

  39. 39.

    O’Connell, R. M. et al. Type I interferon production enhances susceptibility to Listeria monocytogenes infection. J. Exp. Med. 200, 437–445 (2004).

    Article  Google Scholar 

  40. 40.

    In, J. et al. Enterohemorrhagic Escherichia coli reduces mucus and intermicrovillar bridges in human stem cell-derived colonoids. Cell. Mol. Gastroenterol. Hepatol. 2, 48–62 (2016).

    Article  Google Scholar 

  41. 41.

    Vinayak, S. et al. Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum. Nature 523, 477–480 (2015).

    CAS  Article  Google Scholar 

  42. 42.

    Blokzijl, F. et al. Tissue-specific mutation accumulation in human adult stem cells during life. Nature 538, 260–264 (2016).

    CAS  Article  Google Scholar 

  43. 43.

    Zhou, R., Gong, A. Y., Eischeid, A. N. & Chen, X. M. miR-27b targets KSRP to coordinate TLR4-mediated epithelial defense against Cryptosporidium parvum infection. PLoS Pathog. 8, e1002702 (2012).

    CAS  Article  Google Scholar 

  44. 44.

    Chen, X. M., Splinter, P. L., O’Hara, S. P. & LaRusso, N. F. A cellular micro-RNA, let-7i, regulates Toll-like receptor 4 expression and contributes to cholangiocyte immune responses against Cryptosporidium parvum infection. J. Biol. Chem. 282, 28929–28938 (2007).

    CAS  Article  Google Scholar 

  45. 45.

    Faas, F. G. et al. Virtual nanoscopy: generation of ultra-large high resolution electron microscopy maps. J. Cell Biol. 198, 457–469 (2012).

    CAS  Article  Google Scholar 

  46. 46.

    Riggs, M. W. et al. Protective monoclonal antibody defines a circumsporozoite-like glycoprotein exoantigen of Cryptosporidium parvum sporozoites and merozoites. J. Immunol. 158, 1787–1795 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Grun, D. et al. Single-cell messenger RNA sequencing reveals rare intestinal cell types. Nature 525, 251–255 (2015).

    Article  Google Scholar 

  48. 48.

    Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    CAS  Article  Google Scholar 

  49. 49.

    Heiges, M. et al. CryptoDB: a Cryptosporidium bioinformatics resource update. Nucleic Acids Res. 34, D419–D422 (2006).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We are grateful to A.A. Arias, J. Puschhof and H. Hu for assistance with mapping of RNA sequencing data; the Franceschi Microscopy and Imaging Center and D.L. Mullendore at Washington State University for TEM preparation and imaging of isolated organoid oocysts; and C. Joo, J. Beumer and O. Kopper for discussions and critical reading of the manuscript. I.H. is the recipient of a VENI grant from the Netherlands Organization for Scientific Research (NWO-ALW, 863.14.002) and was supported by Marie Curie fellowships from the European Commission (Proposal 330571 FP7-PEOPLE-2012-IIF). D.D. is the recipient of a VENI grant from the Netherlands Organization for Scientific Research (NWO-ALW, 016.Veni.171.015). The research leading to these results has received funding from the European Research Council under ERC Advanced Grant Agreement no. 67013 and from NIH NIAIH under R21 AT009174. This work is part of the Oncode Institute, which is partly financed by the Dutch Cancer Society and was funded by a grant from the Dutch Cancer Society.

Author information

Affiliations

Authors

Contributions

I.H. designed, performed and analysed the experiments and wrote the manuscript. D.D. designed and performed the experiments and helped analyse the data. D.A.S. and M.W.R. performed the mouse experiments. N.I. and P.J.P. performed the TEM experiments. B.A. helped to perform RNA sequencing and analyse the data. N.S. helped in the experiments with lung organoids and immunofluorescence microscopy imaging. K.E.B. helped with the qPCR experiments. R.O. performed oocyst isolation from organoids. G.B. and R.O. performed gene ontology-term analysis of C. parvum genes. A.P.A.H. and R.J.R.W. helped with SEM experiments. R.O. and M.W.R. analysed SEM and TEM data. H.C. and R.O. supervised the project and H.C. wrote the manuscript. All of the authors commented on the manuscript.

Corresponding authors

Correspondence to Roberta O’Connor or Hans Clevers.

Ethics declarations

Competing interests

N.S. and H.C. are inventors on patents/patent applications related to organoid technology.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–7.

Reporting Summary

Supplementary Table 1

Differentially expressed genes in lung and SI organoids at each time point.

Supplementary Table 2

Differentially expressed C. parvum genes between 24- and 72-hour post-injection in lung and SI organoids.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Heo, I., Dutta, D., Schaefer, D.A. et al. Modelling Cryptosporidium infection in human small intestinal and lung organoids. Nat Microbiol 3, 814–823 (2018). https://doi.org/10.1038/s41564-018-0177-8

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing