Knockout of liver fluke granulin, Ov-grn-1, impedes malignant transformation during chronic infection with Opisthorchis viverrini

Abstract

Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor for cholangiocarcinoma (CCA; bile duct cancer) in the Lower Mekong River Basin countries including Thailand, Lao PDR, Vietnam, Myanmar and Cambodia. Using gene edited liver flukes in a hamster model of CCA involving concurrent infection and administration of sub-carcinogenic levels of nitrosamine, we explored the role of the fluke granulin-like growth factor Ov-GRN-1 in malignancy. We produced programmed gene knockout flukes (ΔOv-grn-1) by delivery of a CRISPR/Cas9/gRNA system by square wave electroporation. Targeted genome sequencing confirmed Cas9-catalyzed mutations in target parasite genes, and the rapid depletion of transcripts and the targeted proteins. Hamsters were infected with gene edited larval parasites and exposed to sub-carcinogenic levels of dimethyl nitrosamine in drinking water. Whereas Ov-grn-1 gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes, less hepatobiliary tract disease manifested during chronic infection with ΔOv-grn-1 worms in comparison to hamsters infected with control parasites. Specifically, immunohistochemical analysis of thin sections of hamster livers revealed markedly less fibrosis surrounding flukes and less global liver fibrosis as a result of infection with ΔOv-grn-1 worms, minimal biliary epithelial cell proliferation, and markedly fewer mutations of TP53 in biliary epithelial cells. Moreover, fewer hamsters developed high grade CCA when infected with ΔOv-grn-1 flukes compared to controls. The clinically-relevant pathophysiological phenotype of the livers of the hamsters confirmed a role for this parasite secreted growth factor in morbidity and malignancy during opisthorchiasis.

Transgenesis in parasitic helminths: a brief history and prospects for the future

“…The mutation resulted in reduced pathology when gene-edited parasites colonised the biliary tract of hamsters [ 20 ]. In a recent preprint, these same investigators appeared to confirm a role for liver fluke granulin in malignant transformation of the biliary tract [ 100 ]. Following upon the findings with VSVG pseudotyped MLV, VSVG pseudotyped HIV-1 (human immunodeficiency virus 1) lentiviral virions was also reported to facilitate CRISPR-based transfection of schistosomes [ 80 , 89 ] and to deliver proviral transgenes into the chromosomes of S. mansoni [ 101 ].…”

Small extracellular vesicles but not microvesicles fromOpisthorchis viverrinipromote cell proliferation in human cholangiocytes

“…For instance, over 300 proteins have been identified in the ES products from O. viverrini (4), one of which is a granulin-like growth factor termed Ov-GRN-1, which drives proliferation of biliary epithelial cells. Ov-GRN-1-induced cell proliferation can be inhibited with antibodies raised to the recombinant protein (12), and moreover, infection of hamsters with Ov-grn-1 knock-out flukes results in reduced biliary fibrosis and cholangiocarcinoma compared to hamsters infected with control flukes (8,13). In addition, we reported that O. viverrini secretes small extracellular vesicles (EVs) of 40-100 nm in size that can be internalized by host cells (3), and that blocking internalization of these EVs using antibodies against a member of the tetraspanin protein family significantly reduced the secretion of pro-inflammatory cytokines including IL-6 (3).…”