Developing novel flukicides: the effect of neurotoxic spider venoms on the parasite, Fasciola hepatica

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Fasciola hepatica has a substantial impact on global livestock productivity. Fasciolosis is listed as a top five disease in both sheep and cattle in UK with annual losses exceeding £38million. In the absence of a commercial vaccine control is dependent on availability of a limited number of anthelmintic drugs, known as flukicides. Resistance to the most effective flukicide, triclabendazole, is widespread and poses a threat to sustainable control. This is compounded by climatic changes driving increased transmission of liver fluke and enhanced survival on pasture of the snail intermediate host and extra-mammalian stages. As a result, farmers are increasing reliant on flukicide control of fasciolosis and development of novel flukicides is a priority. Spider venom toxins (SVTs) have been identified as potential biopesticides for a range of insect pests. We have established proof of principle that, in vitro, juvenile F. hepatica are susceptible to venom derived invertebrate-specific neurotoxins that are known to target invertebrate ligand and voltage-gated ion channels. Recently, we have determined that liver fluke have a rich cadre of ligand-gated ion channels, voltage-gated potassium channels and voltage-gated calcium channel subunits; it is notable that most families are upregulated in juvenile fluke. Our working hypothesis is that SVTs act on liver fluke ion channels and have potential as next generation flukicides. The aim of this project is to evaluate the potential of three neurotoxins and interrogate mechanisms underlying their effect in F. hepatica. The objectives of the project are:1. To develop standardised in vitro toxicity assays and produce a panel of recombinant SVT proteins that will be tested using this optimised assay. We have access to a well-defined resource of triclabendazole-susceptible and -resistant fluke isolates.2. To generate 3D tertiary structure predictions of the recombinant proteins using AlphaFold2 (deep learning-based structure prediction) and integrate these data with our toxicity assays, to promote rational design of recombinant toxins.3. To evaluate the stability of recombinant proteins to proteolytic breakdown by fluke gut and secretory enzymes e.g cysteine proteases.4. Using in vitro cultures, investigate how the toxins reach their target tissue, via the gut or outer tegument and identify toxin targets using confocal scanning laser microscopy and in situ hybridisation.5. Once targets have been identified, to establish functionality, gene silencing (RNA interference) will be used to interrogate target-engagement and inform mechanisms of action.