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

线虫种类繁多，分布广泛。一些线虫是寄生虫，会感染植物并导致农作物损失，例如沙氏异形菌，它感染包括甜菜和西兰花在内的200多种植物。这些和其他植物寄生线虫每年造成数十亿磅的作物损失。其他线虫则被认为是“有益的”，例如猫斯氏线虫，它被用于有机园艺，因为它可以在害虫生活的特定细菌的帮助下杀死害虫。一旦杀死了一只昆虫，细菌还会通过产生化学物质来阻止捕食者和竞争对手，包括其他线虫。在这项提议中，我们将利用使斯泰纳线虫具有杀灭能力的细菌来寻找天然的抗线虫化合物，这些化合物可能被用来瞄准损害农作物甚至感染牲畜或人类的寄生线虫。科学家已经研究了斯捷纳线虫相关细菌制造的天然产品，但这些产品是复杂混合物的一部分(或者在实验室条件下根本不生产)，这可能会使识别活性成分变得困难。我们将使用两项新技术，这两项技术将使我们有更好的机会找到活性成分。第一项技术是由我们的合作者开发的。他们对细菌进行了改造，首先移除了一个启动子(一种基因开关)，使细菌无法产生任何致命的化合物。然后，在负责产生单一化合物的基因簇前面一次添加一个特定的启动子，然后在不受通常产生的其他化合物的干扰的情况下测试该化合物的活性。下一个挑战是测试这些化合物的生物活性。我们最近开发了第二项新技术，使这项工作更有效率。我们使用了百万像素的摄像头阵列，可以同时录制数百个样本的视频。每个样本将包含一种细菌提取物，用于与线虫一起测试。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。在以前的工作中，我们发现已知具有不同生物靶点的化合物会导致明显不同的行为变化。因此，我们将能够检测出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一种活性。一旦我们测试了所有细菌化合物，我们就会选择那些对行为或发育有不同影响的有效化合物。强效化合物是最有可能是有用的抗寄生虫化合物。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新实验发现已知化合物的“重新发现”问题。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，并将更大数量的化合物发送给我们在先正达的项目合作伙伴。然后，先正达的科学家们将利用他们的设备，通过遗传和生化方法来确定这些化合物在蠕虫中的作用方式。在完成拟议的工作后，我们将发现对线虫具有活性的天然化合物，解决它们的结构，并找到它们的目标。总而言之，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，最终可能会减少作物损失。这项工作也将有助于对斯捷纳内马生态感兴趣的科学家，并让他们深入了解它们如何完成其非凡的生命周期。

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.