1. Microbiology and Infectious Disease
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Programmed knockout mutation of liver fluke granulin attenuates virulence of infection-induced hepatobiliary morbidity

  1. Patpicha Arunsan
  2. Wannaporn Ittiprasert
  3. Michael J Smout
  4. Christina J Cochran
  5. Victoria H Mann
  6. Sujittra Chaiyadet
  7. Shannon E Karinshak
  8. Banchob Sripa
  9. Neil David Young
  10. Javier Sotillo
  11. Alex Loukas  Is a corresponding author
  12. Paul J Brindley  Is a corresponding author
  13. Thewarach Laha  Is a corresponding author
  1. Khon Kaen University, Thailand
  2. George Washington University, United States
  3. James Cook University, Australia
  4. The University of Melbourne, Australia
Research Article
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Cite this article as: eLife 2019;8:e41463 doi: 10.7554/eLife.41463

Abstract

Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor for cholangiocarcinoma (CCA) in the Lower Mekong River Basin countries including Thailand, Lao PDR, Vietnam and Cambodia. We exploited this link to explore the role of the secreted growth factor termed liver fluke granulin (Ov-grn-1) in pre-malignant lesions by undertaking programmed knockout of the Ov-grn-1 gene from the liver fluke genome. Deep sequencing of amplicon libraries from genomic DNA of gene-edited parasites revealed Cas9-catalyzed mutations within Ov-grn-1. Gene editing resulted in rapid depletion of Ov-grn-1 transcripts and the encoded Ov-grn-1 protein. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes, but the infection resulted in reduced pathology as evidenced by attenuated biliary hyperplasia and fibrosis. Not only does this report pioneer programmed gene-editing in parasitic flatworms, but the striking, clinically-relevant pathophysiological phenotype confirms the role for Ov-grn-1 in virulence morbidity during opisthorchiasis.

Article and author information

Author details

  1. Patpicha Arunsan

    Faculty of Medicine, Department of Parasitology, Khon Kaen University, Khon Kaen, Thailand
    Competing interests
    The authors declare that no competing interests exist.
  2. Wannaporn Ittiprasert

    Department of Microbiology, Immunology and Tropical Medicine, George Washington University, Washington DC, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Michael J Smout

    Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6937-0112
  4. Christina J Cochran

    Department of Microbiology, Immunology and Tropical Medicine, George Washington University, Washington DC, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Victoria H Mann

    Department of Microbiology, Immunology and Tropical Medicine, George Washington University, Washington DC, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Sujittra Chaiyadet

    Faculty of Medicine, Department of Parasitology, Khon Kaen University, Khon Kaen, Thailand
    Competing interests
    The authors declare that no competing interests exist.
  7. Shannon E Karinshak

    Department of Microbiology, Immunology and Tropical Medicine, George Washington University, Washington DC, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2079-0973
  8. Banchob Sripa

    Faculty of Medicine, Department of Pathology, Khon Kaen University, Khon Kaen, Thailand
    Competing interests
    The authors declare that no competing interests exist.
  9. Neil David Young

    Faculty of Veterinaryand Agricultural Sciences, The University of Melbourne, Parkville, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8756-229X
  10. Javier Sotillo

    Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1443-7233
  11. Alex Loukas

    Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, Australia
    For correspondence
    alex.loukas@jcu.edu.au
    Competing interests
    The authors declare that no competing interests exist.
  12. Paul J Brindley

    Department of Microbiology, Immunology and Tropical Medicine, George Washington University, Washington DC, United States
    For correspondence
    pbrindley@gwu.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1765-0002
  13. Thewarach Laha

    Faculty of Medicine, Department of Parasitology, Khon Kaen University, Khon Kaen, Thailand
    For correspondence
    thewa_la@kku.ac.th
    Competing interests
    The authors declare that no competing interests exist.

Funding

National Cancer Institute (R01CA164719)

  • Paul J Brindley

Thailand Research Fund (PHD/0111/2557)

  • Thewarach Laha

Wellcome (107475/Z/15/Z)

  • Paul J Brindley

National Health and Medical Research Council (APP1085309)

  • Alex Loukas

Royal Golden Jubilee PhD Program, Thailand (PHD/0111/2557)

  • Patpicha Arunsan

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: The Animal Ethics Committee of Khon Kaen University approved the study, approval number ACUC-KKU-61/60, which adhered to standard guidelines of the National Research Council of Thailand for the Ethics of Animal Experimentation.

Reviewing Editor

  1. James B Lok, University of Pennsylvania, United States

Publication history

  1. Received: August 27, 2018
  2. Accepted: December 12, 2018
  3. Accepted Manuscript published: January 15, 2019 (version 1)
  4. Version of Record published: January 31, 2019 (version 2)

Copyright

© 2019, Arunsan et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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