LIVER FLUKE MOTOR FUNCTION AND PARASITE CONTROL: EXPLOITING A 'TARGET VALIDATION TOOLBOX' AS A DRUG SCREEN-INTERFACE FOR FLUKICIDE DISCOVERY

片形吸虫是一种寄生虫，也被称为肝吸虫，它会引起一种广泛的动物疾病——片形吸虫病。片形吸虫病对动物健康和福利有着深远的负面影响，破坏了我们的粮食安全，给食品生产商（农民）和食品加工商造成重大损失，据估计全球每年损失约30亿美元，英国每年损失约3亿英镑。在英国，肝吸虫的主要影响是农业，农场层面的损失是巨大的，据估计，2011年每只感染动物的损失为25-30英镑；在屠宰场由于肝吸虫引起的肝脏谴责在牛中占26%，在羊中占6%。在英国，与片形吸虫病有关的一个主要问题是，气候变化正在导致片形吸虫病爆发的风险增加。事实上，对英国肝吸虫患病率变化的估计预测，到2050年，英国部分地区的片吸虫病风险将达到前所未有的水平。目前，还没有针对肝吸虫的疫苗，因此农民依赖药物管理。用于控制肝吸虫的主要药物（杀虫剂）是三氯苄唑（TCBZ），因为它是唯一一种对成虫（生活在胆管中）和幼虫（被植物包裹吞下后，从肠道通过肝脏迁移到胆管，在此过程中造成很大损害）都有显著疗效的药物。过度依赖TCBZ控制肝吸虫已经导致耐药性，澳大利亚和整个欧洲都有报道。事实上，在缺乏新药物类别或疫苗的情况下，TCBZ耐药性威胁到英国一些地区畜牧业的可持续性。显然，迫切需要新的药物来控制肝吸虫。大多数用于控制蠕虫寄生虫的药物都是通过破坏它们的神经和肌肉协同工作和协调正常行为的能力来实现的。换句话说，最好的药物会破坏寄生虫的正常运动功能。运动功能的破坏会阻止幼年吸虫完成其破坏性的迁移，也会阻止成年吸虫附着在宿主的胆管上，进食和繁殖，从而迅速导致蠕虫死亡。因此，更好地了解肝吸虫运动功能将为新药/杀虫剂的发现工作提供更明智的方法。最近与肝吸虫相关的几个发展为这个项目播下了种子。首先，肝吸虫基因组序列几乎完整，基因组数据将包括其运动功能的关键信息。为了利用这些数据，我们需要能够从基因组序列中确定哪些运动功能基因/蛋白质对它们的生存至关重要。这里的一个关键进展是我们最近的发现，肝吸虫是可以反向遗传的，这种技术允许我们在蠕虫中选择一个基因并将其关闭（或使其沉默）。我们现在已经在肝吸虫中优化了这项技术，这样我们就可以沉默蠕虫的任何基因。基因沉默后，行为正常的蠕虫表明该基因不是良好的药物靶标，而表现出异常行为或死亡的蠕虫则与已被证实为良好药物靶标的基因有关。通过使用基因组序列来识别与吸虫运动功能相关的基因，然后依次沉默这些基因，使用反向遗传学，并监测这对蠕虫生物学的影响，我们将创建一个经过验证的药物靶点管道。关键是能够利用这些有效的目标快速控制肝吸虫。为此目的，该项目涉及一个工业合作者的支持，该合作者将在其药物筛选方案中采用经过验证的药物靶点，以促进发现和开发新的杀流剂。通过这种方式，快速利用肝吸虫基因组和生物学的基础研究来帮助发现控制肝吸虫的新药。

Fasciola species of parasitic worms, also known as liver fluke, cause a widespread disease of animals called fasciolosis. Fasciolosis has a profound, negative influence on animal health and welfare, undermining our food security and causing major losses to both food producers (farmers) and food processors estimated at ~$US3 billion/year worldwide and at ~£300 million/year in the UK. In the UK, the major impact of liver fluke is on agriculture, where losses at farm level are dramatic and were estimated to be £25-30 per infected animal in 2011; liver condemnation at abattoir due to liver fluke is 26% in cattle and 6% in sheep. A major concern relating to fasciolosis in the UK is the fact that climate change is causing an increasing risk of fasciolosis outbreaks. Indeed, estimates of changing liver fluke prevalence in the UK have forecast unprecedented levels of fasciolosis risk in parts of the UK by 2050. Currently, there are no vaccines against liver fluke such that farmers rely on the administration of drugs. The main drug (flukicide) used to control liver fluke is triclabendazole (TCBZ) as it is the only drug with significant efficacy against both the adult worms (which lives in the bile ducts) and juveniles (which, after being swallowed encysted on vegetation, migrate from the intestine through the liver to the bile ducts, causing much damage in the process). The over-reliance on TCBZ for liver fluke control has led to drug-resistance, which has been reported in Australia and across Europe. Indeed, TCBZ resistance in the absence of new drug classes or vaccines threatens the sustainability of livestock farming in some UK regions. Clearly, there is a pressing need for new drugs to control liver fluke. Most of the drugs which are used to control worm parasites do so by disrupting the ability of their nerves and muscle to work together and coordinate normal behaviour. In other words, the best drugs disrupt normal parasite motor function. The disruption of motor function would prevent juvenile fluke from completing their damaging migration and would prevent adult fluke from attaching to the host bile ducts, feeding and reproducing, quickly resulting in worm death. Therefore, a better understanding of liver fluke motor function will allow more informed approaches to new drug / flukicide discovery efforts.Several recent developments associated with liver fluke have seeded this project. First, the liver fluke genome sequence is almost complete and data from the genome will include key information on their motor function. To exploit these data, we need to be able to identify which of the motor function genes / proteins identified from the genome sequence are critical to their survival. A key development here is our recent discovery that liver fluke are amenable to reverse genetics, a technology which allows us to select a gene in the worm and to switch it off (or silence it). We have now optimized this technology in liver fluke such that we can silence any gene from the worm. After gene silencing, a worm which behaves normally would indicate that the gene is not a good drug target whereas one which shows aberrant behaviour or dies would associate with a gene that has been validated as a good drug target. By using the genome sequence to identify genes involved in motor function in fluke and then by silencing these genes in turn, using reverse genetics, and monitoring the effect that this has on worm biology, we will create a pipeline of validated drug targets. Critical here is the ability to exploit these validated targets quickly for liver fluke control. To this end, this project involves the support of an industrial collaborator who will adopt validated drug targets into their drug screening programmes to facilitate the discovery and development of new flukicides. In this way, basic research on the liver fluke genome and biology is rapidly used to help discover new drugs for liver fluke control.