Transcriptome analyses across tissues in the liver fluke Fasciola hepatica – from gene expression to drug target characterization

Abstract

Fasciolosis is a food-borne trematode infection caused by the liver fluke Fasciola hepatica and related species. The disease affects human and animal health worldwide and causes considerable economic losses in the global livestock industry. The limited number of therapeutic options and the increasing resistance to anthelminthics complicate disease control and highlight the need for novel anthelmintics or a vaccine. The development of new anthelmintic strategies, however, requires a deeper understanding of fluke biology, particularly regarding organ function and organ-specific gene expression. Technological advancements in transcriptomics technologies have opened up new avenues to study the transcriptional landscape of multicellular parasites. Spatial transcriptomics (ST) enables the visualization of the entire transcriptome of a tissue section in 2D, while preserving the original morphological context. By applying the Visium Spatial Gene Expression Solution (10x Genomics) to adult F. hepatica, over 9,000 genes were captured and eight different tissues were characterized, including the intestine, integument, and reproductive organs. Gene expression profiles and marker genes were identified for each tissue and subsequently validated through in situ hybridization. In addition, a Gene Ontology (GO) enrichment analysis was performed, revealing characteristic biological processes and molecular functions associated with each tissue. The gene expression map further uncovered the tissue-specific expression of drug targets (β-tubulins), vaccine candidates (Ly6 proteins) as well as drug resistance genes (ABC transporters, glutathione S-transferases), indicating tissue-specific biological functions. Finally, the spatial expression data was combined with a database of bioactive molecules, which identified genes in the tegument and intestine that showed homology to targets of approved drugs and drugs in clinical trials. This approach led to the identification of a tegumental protein kinase C beta, for which small-molecule targeting resulted in parasite death. The further course of the work focussed on the parasite’s intestine, a tissue that, like the tegument, is vital for parasite survival. The transcription factor hepatocyte nuclear factor 4 (HNF4) is a highly conserved regulator of metabolic homeostasis and cellular differentiation of endodermal organs such as the liver, gut, and pancreas. However, its role in liver fluke biology was unknown. Using the spatial transcriptome and in situ hybridization, hnf4 expression was localized in the parasite’s gastrodermis. Functional studies utilizing RNA interference (RNAi) and RNA sequencing in immature parasites revealed that HNF4 was essential for the maintenance of gut-associated gene expression, particularly genes encoding proteases such as cathepsins and legumain. RNAi-mediated knockdown of hnf4 expression led to a significant reduction in worm viability in vitro and caused structural disruption of the intestine. Treatment with a commercial small-molecule inhibitor of HNF4, BI6015, had similar effects, although the transcriptomics data indicated that the inhibitor was not HNF4-specific. Further research on the molecular mechanisms by which HNF4 regulates gut biology may identify key pathways essential for parasite survival. Overall, this work provides the first spatial transcriptome of a parasitic flatworm and demonstrates how spatial transcriptomics can advance the understanding of multicellular parasites. The identification of tissue-specific transcripts proofed to be a valuable tool for both basic research and the discovery of new drug targets.