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

肝片吸虫对全球畜牧业生产有重大影响。在英国，肝片吸虫病被列为绵羊和牛的前五大疾病，年损失超过3800万GB。在缺乏商业疫苗的情况下，控制依赖于数量有限的驱虫药，即所谓的杀线虫剂。对最有效的杀线虫剂三氯苯达唑的抗药性普遍存在，并对可持续控制构成威胁。气候变化加剧了肝吸虫的传播，提高了蜗牛中间宿主和非哺乳动物阶段在牧场上的存活率。因此，农民越来越依赖杀吸虫剂控制肝片吸虫病，开发新型杀吸虫剂是当务之急。蜘蛛毒素(SVT)已被确定为一系列害虫的潜在生物杀虫剂。我们已经建立了原理证据，在体外，幼年肝片吸虫对毒液来源的无脊椎动物特异性神经毒素敏感，这些毒素已知以无脊椎动物配体和电压门控离子通道为靶标。最近，我们发现肝吸虫具有丰富的配体门控性离子通道、电压门控性钾通道和电压门控性钙通道亚基；值得注意的是，大多数家族在幼年吸虫体内表达上调。我们的工作假设是，SVT作用于肝吸虫离子通道，有可能成为下一代杀线虫药物。这个项目的目的是评估三种神经毒素的潜力，并询问它们在肝片吸虫中的作用机制。该项目的目标是：1.开发标准化的体外毒性测试，并生产一组重组SVT蛋白，将使用这种优化的测试方法进行测试。我们有一个明确的三氯苯咪唑敏感和耐药吸虫分离株资源。使用AlphaFold2(基于深度学习的结构预测)生成重组蛋白的三维三级结构预测，并将这些数据与我们的毒性测试相结合，以促进重组毒素的合理设计。评价重组蛋白对吸虫肠道和半胱氨酸蛋白酶等分泌酶降解的稳定性。利用体外培养，研究毒素如何通过肠道或外皮到达目标组织，并使用共聚焦扫描激光显微镜和原位杂交识别毒素靶点。一旦确定了靶点，为了建立功能，基因沉默(RNA干扰)将被用来询问靶点接触并告知行动机制。

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.