Engineering biopesticides

Management of agricultural pesticides in the USA and elsewhere is becoming increasingly complicated due to the evolution of insect resistance to classical chemical pesticides and growing community awareness of the environmental damage caused by many agrochemicals. This has necessitated the development of “environmentally friendly” strategies to combat highly resistant insect species. The most promising of these new strategies appears to be the production of transgenic plants or recombinant baculoviruses that express insect-specific peptide toxins. The overriding aim of ongoing work in this laboratory is to isolate and structurally and functionally characterize novel insecticidal neurotoxins that can be used as biopesticides in their own right or as leads for the design of insecticidal substances.

Our work thus far has focused on the isolation and characterization of peptide toxins from the venom of the Australian funnel-web spider. This spider is possibly the most dangerous in the world, having caused numerous deaths in Australia prior to introduction of an antivenom in 1980. However, it has several features that recommend it as a potential source of useful insecticidal compounds. First, funnel-web spiders are mygalomorphs, or primitive spiders. Because mygalomorphs were present before the evolution of flying insects, they do not spin silken snares for prey capture. Rather, mygalomorphs rely heavily on their venom and physical bulk to immobilize prey (insects and other invertebrates). Second, Australian funnel-web spiders have long lifespans; females in the wild can live for 10-20 years and can be kept in captivity for at least five years. In contrast, modern spiders (araneomorphs) rarely live for more than two years. When agitated, the spiders become aggressive and venom can be aspirated from the fang tips into a glass pipette without the need for electrical stimulation. The combination of long lifespan and facile milkings enables sufficient venom to be obtained for detailed biochemical and biophysical characterization of individual venom components.

We have already isolated over a dozen novel insecticidal neurotoxins from the venom of this spider, many of which appear to be promising leads for the development of biopesticides. The three-dimensional structures of these toxins have revealed some surprising features, such as a functionally critical vicinal disulfide bridge in one family of toxins. Recently, we isolated a remarkably potent inhibitor of insect calcium channels that has more than 1000-fold selectivity for invertebrate versus vertebrate calcium channels.

Ongoing work involves thorough functional and structural analysis of these toxins in order to identify key functional epitopes. We have developed recombinant expression systems for several of these toxins, not just to aid structure-function analyses, but also as the first step towards the development of transgenic plants and baculoviruses that express these toxins. We have also implemented a novel genetic screen in Drosophila for identifying the molecular targets of these toxins.

Relevant papers

1. Wang, X., Connor, M., Smith, R., Maciejewski, M.W., Howden, M.E.H., Nicholson, G.M., Christie, M.J., and King, G.F. (2000) Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bridge.
   Nature Structural Biology 7, 505-513. [see report in New Scientist]
   
   
2. Fletcher, J.I., Smith, R., O'Donoghue, S.I.,Nilges, M., Connor, M., Howden, M.E.H., Christie, M.J. & King, G.F. (1997) The structure of a novel insecticidal neurotoxin, omega-atracotoxin-HV1, from the venom of an Australian funnel web spider. Nature Structural Biology 4, 559-566.
   
   
3. Fletcher, J.I., Chapman, B.E., Mackay, J.P., Howden, M.E.H., and King, G.F. (1997) The structure of versutoxin (delta-atracotoxin-Hv1) provides insights into the binding of site-3 neurotoxins to the voltage-gated sodium channel. Structure 5, 1525-1535.