Programmed knockout mutation of liver fluke granulin attenuates virulence of infection-induced hepatobiliary morbidity
Patpicha Arunsan,
Wannaporn Ittiprasert,
Michael J Smout,
Christina J Cochran,
Victoria H Mann,
Sujittra Chaiyadet,
Shannon E Karinshak,
Banchob Sripa,
Neil David Young,
Javier Sotillo,
Alex Loukas,
Paul J Brindley,
Thewarach Laha
Affiliations
Patpicha Arunsan
Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Department of Microbiology, Immunology and Tropical Medicine, George Washington University, Washington DC, United States; Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington DC, United States
Department of Microbiology, Immunology and Tropical Medicine, George Washington University, Washington DC, United States; Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington DC, United States
Centre for Molecular Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, Australia
Christina J Cochran
Department of Microbiology, Immunology and Tropical Medicine, George Washington University, Washington DC, United States; Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington DC, United States
Victoria H Mann
Department of Microbiology, Immunology and Tropical Medicine, George Washington University, Washington DC, United States; Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington DC, United States
Sujittra Chaiyadet
Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand
Department of Microbiology, Immunology and Tropical Medicine, George Washington University, Washington DC, United States; Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington DC, United States
Banchob Sripa
Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand
Department of Microbiology, Immunology and Tropical Medicine, George Washington University, Washington DC, United States; Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington DC, United States
Thewarach Laha
Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand
Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2012) 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 CRISPR/Cas9 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 also the striking, clinically-relevant pathophysiological phenotype confirms the role for Ov-GRN-1 in virulence morbidity during opisthorchiasis.
