Discovering nematicides by phenotypic screening of bacterial natural products in the nematode worm C. elegans

通过在线虫中细菌天然产物的表型筛选发现杀线虫剂。

• 批准号: BB/X007707/1
• 负责人: Andre Brown
• 金额: $ 65.5万
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX007707%2F1
• 关键词: Discovering; nematicides; phenotypic; screening; bacterial

Nematode worms are diverse and widespread. Some nematodes are parasites that infect plants and cause crop loss, such as Heterodera schachtii which infects over 200 plants including sugar beets and broccoli. These and other plant parasitic nematodes cause billions of pounds of crop damage every year. Other nematodes are considered 'beneficial', such as Steinernema feltiae which is used in organic gardening because it kills insect pests with the help of specific bacteria they live with. Once they have killed an insect, the bacteria also help by producing chemicals that deter predators and competitors including other nematodes. In this proposal, we will harness the bacteria that give Steinernema their killing ability to search for natural anti-nematode compounds that might be used to target the parasitic nematodes that damage crops or even those that infect livestock or humans.Scientists have studied the natural products made by Steinernema-associated bacteria already, but the products are part of complex mixtures (or are not produced at all in lab conditions) which can make it hard to identify the active components. We will use two new technologies that will give us a better chance of finding the active components. The first technology was developed by our collaborators. They engineered the bacteria by first removing a promoter (a genetic 'on switch') that renders the bacteria unable to produce any of their killer compounds. Then, specific promoters are added one at a time in front of gene clusters that are responsible for producing a single compound which can then be tested for activity without interference from the other compounds that would normally be produced.The next challenge is to test the compounds for bioactivity. We have recently developed the second new technology that makes this more efficient. We have used arrays of megapixel cameras that can record videos from hundreds of samples at the same time. Each sample will contain a single bacterial extract for testing along with nematode worms. We then use computational analysis to measure different aspects of how the worms grow and move. In previous work, we found that compounds that were known to have different biological targets caused recognisably different behaviour changes. We will therefore be able to detect bacterial compounds with diverse actions without having to do a separate experiment to detect each kind of activity.Once we have tested all of the bacterial compounds, we will select compounds that are potent and have diverse effects on behaviour or development. Potent compounds are the most likely to be useful anti-parasitic compounds. We will select compounds with diverse effects on behaviour and development because that will minimise the problem of 'rediscovery' in which a new experiment finds already-known compounds. For these priority compounds, our collaborators will use chemical methods to identify the structure of the compounds and send larger quantities to our project partners at Syngenta. Scientists at Syngenta will then use their facilities to determine how the compounds work in worms using both genetic and biochemical methods.Upon completion of the proposed work, we will have discovered natural compounds that are active against nematodes, solved their structure, and found their targets. Together, this information provides a strong starting point for the further development of these compounds as nematicides that might eventually reduce crop loss. The work will also be useful for scientists interested in the ecology of Steinernema and give insight into how they complete their remarkable lifecycle.

线虫种类繁多，分布广泛。有些线虫是感染植物并造成作物损失的寄生虫，例如沙氏异源线虫（Heterodera schachtii）感染200多种植物，包括甜菜和西兰花。这些和其他植物寄生线虫每年造成数十亿磅的作物损失。其他的线虫被认为是“有益的”，比如在有机园艺中使用的feltiae线虫，因为它在与害虫共生的特定细菌的帮助下杀死害虫。一旦它们杀死了一只昆虫，这些细菌也会通过产生化学物质来阻止捕食者和包括其他线虫在内的竞争对手。在这个提议中，我们将利用赋予斯坦纳线虫杀死能力的细菌来寻找天然的抗线虫化合物，这些化合物可能用于靶向破坏作物甚至感染牲畜或人类的寄生线虫。科学家们已经研究了与斯坦纳氏菌相关的细菌产生的天然产物，但是这些产物是复杂混合物的一部分（或者根本不是在实验室条件下产生的），这使得很难识别有效成分。我们将使用两种新技术，这将使我们有更好的机会找到有效成分。第一项技术是由我们的合作者开发的。他们首先去除一个启动子（一种基因“开启开关”）来改造细菌，使细菌无法产生任何杀伤化合物。然后，在负责产生单一化合物的基因簇前一次添加一个特定的启动子，然后可以在不受通常会产生的其他化合物干扰的情况下测试其活性。下一个挑战是测试化合物的生物活性。我们最近开发了第二项新技术，可以提高效率。我们使用了百万像素的相机阵列，可以同时记录数百个样本的视频。每个样品将包含一种细菌提取物和线虫进行测试。然后，我们使用计算分析来测量蠕虫如何生长和移动的不同方面。在之前的工作中，我们发现已知具有不同生物靶点的化合物会引起明显不同的行为变化。因此，我们将能够检测具有不同作用的细菌化合物，而不必进行单独的实验来检测每种活性。一旦我们测试了所有的细菌化合物，我们将选择有效的化合物，对行为或发育有不同的影响。强效化合物最有可能是有效的抗寄生虫化合物。我们将选择对行为和发育有不同影响的化合物，因为这将最大限度地减少“重新发现”的问题，即在新的实验中发现已知的化合物。对于这些优先化合物，我们的合作者将使用化学方法来确定化合物的结构，并将大量的化合物发送给我们在先正达的项目合作伙伴。然后，先正达的科学家将利用他们的设备，通过遗传和生化方法来确定这些化合物如何在蠕虫体内起作用。在完成这项工作后，我们将发现对线虫有活性的天然化合物，解决它们的结构，并找到它们的靶点。总之，这些信息为进一步开发这些化合物作为杀线虫剂提供了一个强有力的起点，最终可能减少作物损失。这项工作也将有助于科学家对steinerma的生态学感兴趣，并深入了解它们如何完成其非凡的生命周期。