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

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.

Nematode parasites cause billions of pounds of crop damage every year, but many existing nematicides are broad-acting toxins and are thus not widely used due to safety concerns. To discover candidate nematicides that may be safer and more targeted, we will screen a library of microbial natural products for behavioural and developmental effects on the nematode worm C. elegans followed by chemical methods to solve their structures and biochemical and genetic methods to identify their nematode targets.

We will focus on compounds produced by Xenorhabdus and Photorhabdus bacteria which are symbionts of entomopathogenic nematodes. They are a promising source of nematicides because there is selective pressure for them to produce compounds that kill their insect hosts and also protect the insect cadaver from non-symbiotic nematodes. Despite this promise, they are underexplored because of the difficulty of isolating specific molecules for testing. To unlock this considerable potential, our collaborators have produced a library of extracts from Xenorhabdus and Photorhabdus that have been engineered with inducible promoters that activate one biosynthetic gene cluster at a time.

We will use a combination of high-content imaging and machine learning methods to identify extracts that produce behavioural or developmental phenotypes. By clustering compounds according to their multidimensional phenotypic effects and comparing them against the effects of known compounds, we will prioritise those that are most likely have novel modes of action. Our collaborators will then solve the priority compounds' structures and share larger quantities for follow-up experiments including in vitro experiments and forward genetic screens to identify the compounds' nematode targets.

The result will be candidate nematicides with diverse modes of action, new nematode targets, and insight into the chemical ecology of entomopathogenic microbes.