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

确定神经毒性蜘蛛毒液对肝片吸虫寄生虫影响的机制。

• 批准号: 2443686
• 金额: --
• 依托单位: University of Liverpool
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2443686
• 关键词: Identifying; mechanism; underlying; effect; neurotoxic

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.

寄生虫肝片吸虫（肝吸虫）是反刍动物疾病的重要原因，也是全球畜牧业生产的主要制约因素。农业和园艺发展委员会和农业中药物的负责任使用最近的一份报告确定，片形吸虫病是英国绵羊和牛的五大疾病之一，估计每年损失至少3800万英镑。肝吸虫的控制几乎完全依赖于使用特定的兽药或杀吸虫剂。市场上的杀吸虫剂数量有限，对其中一种最有效的三氯苯达唑的耐药性在全球范围内广泛存在。肝吸虫的生命周期依赖于软体动物的中间宿主，Galba truncatula。两种G.该寄生虫在哺乳动物以外的阶段的生长依赖于温和的温度和降雨。随着气候的变化，肝吸虫预计将变得更加广泛和流行。农民越来越依赖于使用杀吸虫剂来控制片形吸虫病，同时，驱虫剂抗性正在成为吸虫控制的主要障碍。在不久的将来，没有出现用于控制片形吸虫病的商业疫苗的前景，因此开发新药对帮助控制疾病至关重要。蜘蛛毒毒素作为一种潜在的生物农药，对一系列害虫的使用有相当大的兴趣，但它们对寄生虫的影响还没有得到很好的研究。在原理研究的证明中，申请人最近表明，在体外，未成熟的F。肝脏对两种神经毒性蜘蛛毒液蛋白敏感，已知这两种蛋白在其它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在F.肝。出于保密/IP原因，我们将它们命名为SVT 1和SVT 2。该项目符合BBSRC农业和粮食安全主题，以提高畜牧业的生产力，健康和福利。我们的工作假设是SVT 1/2作用于肝吸虫电压门控离子通道，并有潜力作为下一代杀吸虫剂。该项目的目标是：1。开发标准化的体外毒性试验，并生产一组将在这些试验中进行检测的重组蛋白。我们已经获得了已知对三氯苯达唑敏感的吸虫分离株的明确样本组。迄今为止的结果表明，敏感分离株和耐药分离株对STV1.2的杀伤力相同。使用体外培养，研究毒素如何通过肠道或外皮层到达其靶组织。我们将评估重组蛋白在吸虫肠酶存在下的稳定性;半胱氨酸蛋白酶在吸虫肠中特别占优势。开采F. hepatica基因组中关于钠和钙门控离子通道的信息。对F. Hepatica基因组表明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作用靶点.一旦靶点被确定，为了建立功能性，基因沉默（RNA干扰）将用于询问靶点接合并告知作用机制。这是一项在合作伙伴3的实验室中得到很好建立的技术。