Programmed knockout mutation of liver fluke granulin attenuates virulence of infection-induced hepatobiliary morbidity
- Khon Kaen University, Thailand
- George Washington University, United States
- James Cook University, Australia
- The University of Melbourne, Australia
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.
Data availability
The Illumina sequencing reads and related project details are available at GenBank: Bioproject PRJNA385864, Biosample SAMN07287348, SRA study SRP110673, accessions SRR5764463-5764618 and SRR8187484-SRR8187487, at https://www.ncbi.nlm.nih.gov/Traces/study/?acc=SRP110673, Bioproject, www.ncbi.nlm.nih.gov/bioproject/PRJNA385864.
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Opisthorchis viverrini Raw sequence readsGenBank, PRJNA385864.
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Sequence: FJ436341.1European Nucleotide Archive, FJ436341.
Article and author information
Author details
Shannon E Karinshak
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.
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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Programmed knockout mutation of liver fluke granulin attenuates virulence of infection-induced hepatobiliary morbidity
eLife 8:e41463.
https://doi.org/10.7554/eLife.41463
Further reading
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- Microbiology and Infectious Disease
The first reports of CRISPR/Cas9 genome editing in flatworms could usher in a new era of research on these dangerous human parasites.
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- Microbiology and Infectious Disease
Saprolegnia parasitica is one of the most virulent oomycete species in freshwater aquatic environments, causing severe saprolegniasis and leading to significant economic losses in the aquaculture industry. Thus far, the prevention and control of saprolegniasis face a shortage of medications. Linalool, a natural antibiotic alternative found in various essential oils, exhibits promising antimicrobial activity against a wide range of pathogens. In this study, the specific role of linalool in protecting S. parasitica infection at both in vitro and in vivo levels was investigated. Linalool showed multifaceted anti-oomycetes potential by both of antimicrobial efficacy and immunomodulatory efficacy. For in vitro test, linalool exhibited strong anti-oomycetes activity and mode of action included: (1) Linalool disrupted the cell membrane of the mycelium, causing the intracellular components leak out; (2) Linalool prohibited ribosome function, thereby inhibiting protein synthesis and ultimately affecting mycelium growth. Surprisingly, meanwhile we found the potential immune protective mechanism of linalool in the in vivo test: (1) Linalool enhanced the complement and coagulation system which in turn activated host immune defense and lysate S. parasitica cells; (2) Linalool promoted wound healing, tissue repair, and phagocytosis to cope with S. parasitica infection; (3) Linalool positively modulated the immune response by increasing the abundance of beneficial Actinobacteriota; (4) Linalool stimulated the production of inflammatory cytokines and chemokines to lyse S. parasitica cells. In all, our findings showed that linalool possessed multifaceted anti-oomycetes potential which would be a promising natural antibiotic alternative to cope with S. parasitica infection in the aquaculture industry.
