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A Novel Method to Silence Genes in Cryptosporidium

  • Alejandro Castellanos-GonzalezEmail author
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Part of the Methods in Molecular Biology book series (MIMB, volume 2052)

Abstract

The parasites of the genus Cryptosporidium are important causes of diarrheal diseases, specifically cryptosporidiosis, worldwide. A major bottleneck for developing drugs and vaccines against cryptosporidiosis is the lack of methods to study gene function in this parasite. Silencing of genes by RNA interference (RNAi) is a powerful method to investigate gene function that has been widely used in the identification of targets for several pathogens. Unfortunately, as Cryptosporidium does not possess the enzymes of the RNAi pathway, its genes cannot be silenced by standard siRNA technology. To circumvent that problem, we have developed a novel strategy to knock down Cryptosporidium genes by reconstituting the effector arm of the siRNA pathway. We have induced silencing of several genes in Cryptosporidium by transfecting parasites with hybrid complexes formed between recombinant human Argonaute (hAgo2) and Cryptosporidium single-stranded RNA (ssRNA). This novel methodology provides an effective strategy to study the role of selected genes in host–parasite interactions, and also can be used to identify potential targets for chemotherapy. The standardized methodology based on this strategy is described in this chapter.

Keywords

Cryptosporidium Gene silencing RNAi 

Notes

Acknowledgments

This work was supported by the Bill & Melinda Gates Foundation grant: OPP1161026 and by the National Institute of Allergy and Infectious Diseases (NIH/NIAID), grant: 5R21AI12627502.

References

  1. 1.
    Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391(6669):806–811.  https://doi.org/10.1038/35888CrossRefPubMedGoogle Scholar
  2. 2.
    Agrawal N, Dasaradhi PV, Mohmmed A, Malhotra P, Bhatnagar RK, Mukherjee SK (2003) RNA interference: biology, mechanism, and applications. Microbiol Mol Biol Rev 67(4):657–685CrossRefGoogle Scholar
  3. 3.
    Rivas FV, Tolia NH, Song JJ, Aragon JP, Liu J, Hannon GJ, Joshua-Tor L (2005) Purified Argonaute2 and an siRNA form recombinant human RISC. Nat Struct Mol Biol 12(4):340–349.  https://doi.org/10.1038/nsmb918CrossRefPubMedGoogle Scholar
  4. 4.
    Batista TM, Marques JT (2011) RNAi pathways in parasitic protists and worms. J Proteome 74(9):1504–1514.  https://doi.org/10.1016/j.jprot.2011.02.032CrossRefGoogle Scholar
  5. 5.
    Kolev NG, Tschudi C, Ullu E (2011) RNA interference in protozoan parasites: achievements and challenges. Eukaryot Cell 10(9):1156–1163.  https://doi.org/10.1128/EC.05114-11CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Lye LF, Owens K, Shi H, Murta SM, Vieira AC, Turco SJ, Tschudi C, Ullu E, Beverley SM (2010) Retention and loss of RNA interference pathways in trypanosomatid protozoans. PLoS Pathog 6(10):e1001161.  https://doi.org/10.1371/journal.ppat.1001161CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Xu P, Widmer G, Wang Y, Ozaki LS, Alves JM, Serrano MG, Puiu D, Manque P, Akiyoshi D, Mackey AJ, Pearson WR, Dear PH, Bankier AT, Peterson DL, Abrahamsen MS, Kapur V, Tzipori S, Buck GA (2004) The genome of Cryptosporidium hominis. Nature 431(7012):1107–1112.  https://doi.org/10.1038/nature02977CrossRefPubMedGoogle Scholar
  8. 8.
    Abrahamsen MS, Templeton TJ, Enomoto S, Abrahante JE, Zhu G, Lancto CA, Deng M, Liu C, Widmer G, Tzipori S, Buck GA, Xu P, Bankier AT, Dear PH, Konfortov BA, Spriggs HF, Iyer L, Anantharaman V, Aravind L, Kapur V (2004) Complete genome sequence of the Apicomplexan, Cryptosporidium parvum. Science 304(5669):441–445.  https://doi.org/10.1126/science.1094786CrossRefPubMedGoogle Scholar
  9. 9.
    Castellanos-Gonzalez A, Perry N, Nava S, White AC Jr (2016) Preassembled single-stranded RNA-Argonaute complexes: a novel method to silence genes in Cryptosporidium. J Infect Dis 213(8):1307–1314.  https://doi.org/10.1093/infdis/jiv588CrossRefPubMedGoogle Scholar
  10. 10.
    Castellanos-Gonzalez A, Sparks H, Nava S, Huang W, Zhang Z, Rivas K, Hulverson MA, Barrett LK, Ojo KK, Fan E, Van Voorhis WC, White AC Jr (2016) A novel calcium-dependent kinase inhibitor, bumped kinase inhibitor 1517, cures cryptosporidiosis in immunosuppressed mice. J Infect Dis 214(12):1850–1855.  https://doi.org/10.1093/infdis/jiw481CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Tilley M, Upton SJ (1994) Both CP15 and CP25 are left as trails behind gliding sporozoites of Cryptosporidium parvum (Apicomplexa). FEMS Microbiol Lett 120(3):275–278CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Infectious Disease Division, Department of Internal MedicineUniversity of Texas Medical BranchGalvestonUSA

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