The dopaminergic signalling system in the liver fluke, Fasciola hepatica
Date
2024-06
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Publisher
Queen University Belfast
Abstract
Diseases caused by parasitic platyhelminths threaten agricultural and human health. Anthelmintic medications are used to treat these parasites, but increasing treatment resistance and climate change make long-term control difficult. This thesis investigated the involvement of the dopamine (DA) signalling system in liver fluke biology, a generally unknown target system in platyhelminths. In addition to classical dopamine signalling components, studies included polypyrimidine tract-binding proteins (PTBs), recently implicated in the redevelopment of DA neurons in mammals. Publically available omics datasets were used to search for dopamine signalling components and PTBs in flatworm parasites, with a focus on the liver fluke, Fasciola hepatica. In silico identification of DA pathway components across platyhelminths, revealed that the essential components of the dopamine signalling machinery are evolutionarily conserved throughout most platyhelminths, with the exception of cestodes and some monogeneans. In silico methods revealed for the first time in platyhelminths the presence of PTBs with some variability in their consensus signature motifs compared to those seen in mammals. Drug bioassays were used to expose the effects of DA and a dopamine reuptake transporter inhibitor on juvenile F. hepatica. Furthermore, functional genomics in the form of RNA interference (RNAi)-induced gene silencing was combined with an in vitro liver fluke culture system to interrogate gene function. The addition of dopamine and inhibition of dopamine reuptake consistently led to altered motility and growth in juvenile F. hepatica. The silencing of a dopamine synthetic enzyme (aromatic dopamine decarboxylase; FhAADC) and a vesicular monoamine transporter (FhVMAT) reduced both the motility and growth of worms. In contrast, dopamine beta hydroxylase (FhDBH) silencing increased both the motility and growth of worms. Combinatorial RNAi of the three putative FhPTBs severely impaired the motility and growth of juvenile worms, and eventually resulted in the death of juvenile worms, inhibiting neoblast-like cell proliferation as well as negatively impacting nervous system development. This validated the potential of dopamine signalling and PTBs as new flukicide targets and encourages a more focused effort on their exploitation as new drug targets for liver fluke control.
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Keywords
5-HT 5-hydroxytryptamine A Alanine AADC Aromatic L-amino acid decarboxylase AADs Amino acetonitrile derivatives AbD Antibody diluent ACh Acetylcholine AChE Acetylcholinesterase ALBZ Albendazole ATP Adenosine triphosphate BA Biogenic Amine BLAST Basic local alignment search tool BSA Bovine serum albumin BTCP N-[1-(2-benzo(b)thiopenyl)cyclohexyl]piperidine BUSCO Benchmarking Universal Single-Copy Orthologs CA Catecholamine cDNA Complementary DNA cg cerebral ganglion CLANs Clustered analysis of sequences algorithm CLORS Clorsulon CLOS Closantel CLSM Confocal laser scanning miscroscopy CNS Central nervous system co Commissure CS Chicken serum Cu Copper DA Dopamine DAPI 4′, 6-diamidino-2-phenylindole dihydrochloride DAT DA reuptake transporter DBH Dopamine beta hydroxylase DEPC Diethyl pyrocarbonate DMF Dimethylformamide DMSO Dimethylsulfoxide DNA Deoxyribonucleic acid DNC Dorsal nerve cord Dopamine 3, 4-dihydroxyphenethylamine dsRNA Double-stranded RNA EdU 5-Ethynyl-2’-deoxyuridine ELISA Enzyme-Linked Immunosorbent Assay EP Epinephrine FEC faecal egg counts FISH Fluorescent in situ hybridization FITC Fluorescein isothiocyanate GABA γ-Aminobutyric Acid GAD Glutamate decarboxylase GAPDH Glyceraldehyde 3-phosphate dehydrogenase GluCl Glutamate-gated Cl- GPCR G-protein coupled receptor hnRNP heterogeneous nuclear HPLC High Performance Liquid Chromatography iGluRs—LGICs ionotropic glutamate receptors IRES Internal ribosomal entry site ISH In situ hybridization KD Knockdown L-AADC L-aromatic amino acid decarboxylase L-DOPA L-3, 4-dihydroxyphenylalanine LGICs ligand-gated ion channels LiCl Lithium chloride LNC Lateral nerve cords MeOH Methanol MFS Major facilitator superfamily mGluRs—GPCRs Metabotropic glutamate receptors MNC Main nerve cords mRNA Messenger RNA N Asparagine NCBI National Centre for Biotechnology Information NE Norepinephrine NEJs Newly excysted juvenile Neo Neomycin phosphotransferase ns Not significant NTD Neglected tropical disease OA Octopamine os oral sucker P-gps P-glycoproteins PAH Phenylalanine hydroxylase PBS Phosphate buffered saline PCP phencyclidine PCR Polymerase chain reaction PFA Paraformaldehyde ph Pharynx PLP Pyridoxal phosphate PNS Peripheral nervous system pre-mRNA pre-messenger RNA PTB Polypyrimidine tract binding proteins qPCR Quantitative PCR RAFOX Rafoxanide RBD RNA binding domains RBM RNA binding MOTIFS RISC RNA-induced silencing complex RNA Ribonucleic acid