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力有重大影响。片形吸虫病被列为英国牛羊的五大疾病之一，每年造成的损失超过3800万英镑。在没有商业疫苗的情况下，控制依赖于有限数量的驱虫药，即杀流剂。对最有效的杀氟剂三氯咪唑的耐药性普遍存在，并对可持续控制构成威胁。气候变化增加了肝吸虫的传播，提高了蜗牛在牧场的中间宿主和非哺乳动物阶段的存活率。因此，农民越来越依赖杀虫剂控制片形虫病，开发新型杀虫剂是当务之急。蜘蛛毒液毒素（SVTs）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明，在体外，幼年肝棘球蚴易受毒液衍生的无脊椎动物特异性神经毒素的影响，这些毒素已知以无脊椎动物配体和电压门控离子通道为目标。最近，我们发现肝吸虫具有丰富的配体门控离子通道、电压门控钾通道和电压门控钙通道亚基；值得注意的是，大多数家庭在幼年吸虫中是上调的。我们的工作假设是svt作用于肝吸虫离子通道，有潜力成为下一代杀虫剂。本项目的目的是评估三种神经毒素的潜力，并询问其在肝梭菌中的作用机制。该项目的目标是：1。开发标准化的体外毒性试验，并生产一组重组SVT蛋白，将使用这种优化的试验进行测试。我们已经获得了定义明确的三氯苯达唑敏感和耐药吸虫分离物资源。利用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试验相结合，以促进重组毒素的合理设计。评价重组蛋白在吸虫肠道和分泌酶（如半胱氨酸蛋白酶）水解分解时的稳定性。使用体外培养，研究毒素如何通过肠道或外被到达目标组织，并使用共聚焦扫描激光显微镜和原位杂交技术识别毒素目标。一旦确定了靶标，为了确定功能，基因沉默（RNA干扰）将用于询问靶标接合并告知作用机制。

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.