Identifying the mechanism underlying the effect of neurotoxic spider venoms on the parasite Fasciola hepatica.

这种寄生虫，肝吸片虫（肝吸虫），是反刍动物疾病的一个重要原因，也是全球畜牧业生产的一个主要制约因素。最近一份来自农业和园艺发展局和农业药品负责任使用的报告指出，片形吸虫病是英国羊和牛的五大疾病之一，估计每年损失至少3800万英镑。控制肝吸虫几乎完全依靠使用特定的兽药或杀虫剂。市场上杀菌剂的数量有限，对最有效的杀菌剂之一三氯咪唑的耐药性在全球范围内普遍存在。肝吸虫的生命周期依赖于一种软体动物中间宿主，斑蝥。这两种寄生虫的超哺乳动物阶段都依赖于温和的温度和降雨。据预测，随着气候的变化，肝吸虫将变得更加广泛和流行。农民越来越依赖于使用杀虫剂来控制片形虫病，与此同时，驱虫抗药性正在成为控制吸虫的主要障碍。在不久的将来，用于控制片形吸虫病的商业化疫苗还没有出现的前景，因此开发新药对帮助控制疾病至关重要。人们对使用蜘蛛毒液毒素作为潜在的生物杀虫剂来对付一系列害虫很感兴趣，但它们对寄生虫的影响还没有得到很好的研究。在原理证明研究中，申请人最近表明，在体外，未成熟的肝原丝胞菌对两种神经毒性蜘蛛毒液蛋白敏感，这两种蛋白已知在其他系统中作用于不同的电压门控离子通道。本项目的目的是研究这些毒素如何在肝梭菌中发挥作用的机制。出于保密/IP原因，我们将其命名为SVT1和SVT2。该项目符合BBSRC农业与粮食安全主题，旨在提高畜牧业的生产力、健康和福利。我们的工作假设是SVT1/2作用于肝吸虫电压门控离子通道，有潜力成为下一代杀流剂。该项目的目标是：1。开发标准化的体外毒性试验，并生产一组重组蛋白，用于这些试验。我们已经获得了对三氯苯达唑敏感的吸虫分离物的明确鉴定小组。迄今为止的结果表明，敏感株和耐药株同样容易被STV1.2杀死。使用体外培养，研究毒素如何通过肠道或外被膜到达目标组织。我们将评估重组蛋白在肠道酶存在下的稳定性；半胱氨酸蛋白酶在吸虫肠道中尤其占优势。挖掘肝梭菌基因组中钠钙门控离子通道的信息。初步分析表明肝螺旋体基因组存在一系列电压门控离子通道，包括钠、钙和钾通道。4. 利用激光共聚焦扫描显微镜和原位杂交技术鉴定毒素的靶点。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。这是一种在合作伙伴3的实验室中建立起来的技术。

The parasite, Fasciola hepatica (liver fluke), is a significant cause of disease in ruminants and is a major constraint on livestock production globally. A recent report from the Agriculture and Horticulture Development Board and Responsible Use of Medicines in Agriculture identified fasciolosis in the top five disease in both sheep and cattle in UK with estimated annual losses of at least £38 million.Control of liver fluke relies almost exclusively on the use of specific veterinary medicines or flukicides. There are a limited number of flukicides on the market and resistance to one of the most effective, triclabendazole, is widespread globally. The liver fluke life cycle relies on a mollusc intermediate host, Galba truncatula. Both G. truncatula and the extra-mammalian stages of the parasite are dependent on mild temperatures and rainfall. Liver fluke is predicted to become more widespread and prevalent as the climate changes. Farmers are becoming more dependent on the use of flukicides to control of fasciolosis and concomitantly, anthelmintic resistance is becoming a major impediment to fluke control. There is no prospect of a commercial vaccine for control of fasciolosis appearing in the near future, so development of novel drugs is vital to help control disease. There is considerable interest in the use of spider venom toxins as potential biopesticides for a range of insect pests but their effect on parasites is not well studied. In proof of principle studies, the applicants have recently shown that, in vitro, immature F. hepatica are susceptible to two neurotoxic spider venom proteins that are known to act in other systems on distinct voltage-gated ion channels. The aim of this project is to investigate the mechanisms underlying how these toxins exert their effect in F. hepatica. For confidentiality /IP reasons, we have named them as SVT1 & SVT2. This project fits into the BBSRC Agriculture and Food Security theme, to improve productivity, health and welfare in the livestock sector.Our working hypothesis is that SVT1/2 act on liver fluke voltage-gated ion channels and have potential as next generation flukicides. The objectives of the project are:1. To develop standardised in vitro toxicity assays and produce a panel of recombinant proteins that will be tested in those assays. We have access to well defined panels of fluke isolates of known susceptibility to triclabendazole. Results to date suggest that both susceptible and resistant isolates are equally susceptible to killing by STV1.2. Using in vitro cultures, investigate how the toxins reach their target tissue, via the gut or outer tegument. We will evaluate the stability of the recombinant proteins in the presence of fluke gut enzymes; cysteine proteases are particularly dominant in the fluke gut.3. To mine the F. hepatica genome for information about sodium and calcium gated ion channels. Preliminary analysis of the F. hepatica genome suggests the presence of a range of voltage-gated ion channels including sodium, calcium and potassium channels. 4. To identify the targets of the toxins using confocal scanning laser microscopy and in situ hybridisation.5. Once the targets have been identified, to establish functionality, gene silencing (RNA interference) will be used to interrogate target-engagement and inform mechanisms of action. This is a technique that is well established in the laboratory of partner 3.