Characterising a critical role for novel glycosphingolipids in the mechanism of action of insecticidal Toxin_10 proteins

Mosquitoes are major carriers of human diseases, including Zika virus, malaria and west Nile fever. There are not many safe insecticides available to kill mosquitoes that are not damaging to the environment or human health. One of the few available is Bin toxin, which is made by a particular type of bacteria called Lysinibacillus sphaericus, which kills mosquito larvae. However, mosquitoes are developing resistance to the toxin. It is vital that we find out how Bin toxin kills mosquitoes so that it can be made more effective or that alternatives can be identified. Not much is known about how Bin toxin kills mosquitoes. We know it binds to a particular protein in the gut of mosquito larvae. Binding of the toxin to this protein causes changes in the cells in the mosquito gut - the toxin is taken into cells of the gut causing large bubbles to form inside them and somehow this leads to death of cells in the gut and then death of the mosquito. Bin toxin has two parts, BinA and BinB, it is BinB that binds to the protein in the mosquito gut but both parts are needed to kill mosquitoes. Both BinA and BinB are part of a family of toxins called Toxin_10. This toxin family are similar in certain ways to proteins that bind sugars, especially sugars which are attached to lipids (fats). Several toxins are already known to us that use their ability to bind (stick to) these sugar modified fats in order to gain entry into cels and then kill their target cells once they are inside. These include the toxins involved in cholera and tetanus. We have evidence that Bin toxin also binds to one of these sugar modified fats, which we call "glycolipids", but we don't know which one yet. We aim to discover exactly which glycolipid Bin toxin binds to, and whether this is important for Bin toxin to be able to cause bubble formation inside cells and kill mosquitoes. We will also discover which exact parts of the Bin toxin proteins are responsible for binding the glycolipid, and whether making alterations in these areas changes the ability of Bin toxin to bind glycolipid or kill mosquitoes, which may help make a more effective insecticide. Other members of the Toxin_10 family that Bin toxin belongs to kill different insects, several of which are economically important agricultural pests. Resistance to current pesticides is also a major problem in agriculture, and there is a pressing need for alternative methods to control these pests. One of these Toxin_10 family members is Cry35, which kills the Western corn root worm, a major problem in maize production in the USA. We will investigate whether Cry35, like Bin toxin, also binds a glycolipid and, if it does, whether this is important for its ability to kill Western corn root worm. This research has the potential to find out how Toxin_10 toxins are killing insects. With this information, we may be able to make them more effective pesticides, thus reducing the spread of mosquito-borne disease and agricultural reliance on chemical pesticides.