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.

We aim to characterise the role of a unique insect glycosphingolipid (GSL) in Bin toxin action. Bin toxin is a two-part toxin of the Toxin_10 (T10) family, comprising BinA and BinB, that kills Culex and Anopheles mosquitoes, carriers of Zika virus and malaria. Bin toxin is the best characterised T10 toxin. It binds a receptor (Cqm1) in the larval midgut, followed by toxin internalisation and formation of large vacuoles inside cells before death. MDCK cells expressing Cqm1 also internalise toxin and form vacuoles, but do not die. We find that mammalian cells with altered GSL synthesis (GM3 synthase null MEF and CHO cells) are more affected by Bin toxin, and that this can be reversed using the GSL synthesis inhibitor miglustat. We hypothesise that Bin toxin binds an unusual GSL in mutant mammalian cells that is similar to an endogenous GSL in the larval mosquito midgut, and that this is important for toxicity. We propose to comprehensively characterise the role of GSL in Bin toxin action. We will identify the GSL to which Bin toxin binds using lipid dot-blots of mammalian and mosquito GSL. We will uncover the structural basis of this interaction by mutating key amino acids in the beta-trefoil carbohydrate binding domains of BinA and BinB, modelling the Bin toxin/GSL interaction and attempting to co-crystallise Bin toxin with the GSL. We will determine roles for intracellular Ca2+ and phosphoinositol signalling in Bin toxin action using genetic and pharmacological modifiers. Finally, we will determine whether GSL are important for Bin toxicity in Culex mosquito larvae by using miglustat to reduce GSL in vivo. The work outlined in this proposal will characterise a completely novel aspect of Bin toxin action, which will help overcome Bin toxin resistance and provide a mechanistic model for other T10 toxins. Key findings will be replicated in Cry35, an important T10 protein in agricultural biotechnology that is a major interest of the industrial partner on this project.

From this basic research grant there are a number of beneficiaries including the scientific community, industrial partners and world health organizations as well as the general public. Expert training of the researcher co-investigator will contribute directly to the local and wider science base, professional training will be ensured through the infrastructure provided by Cardiff University's world-class research and world-leading career development programmes. The mechanism of action of the toxin family we are researching is vital from the perspective of increasing sustainable agricultural yield and food safety and is demonstrably relevant to disease through the connections to malaria, Zika and West Nile fever transmission. The results that emerge from this study will be of clear interest to all of those communities. In general a better understanding of toxin function will lead to potential wider development of the Toxin_10 family as biologically safe insecticidal agents and an improvement in our knowledge of fundamental cellular processes (endocytosis) as well as potential development of new cell biological imaging tools (e.g. cancer cell markers). This project is clearly of interest to agrochemical companies. This is evidenced by the involvement of Dow AgroScience on this proposal as well as the clear roles of multiple companies in the field developing these toxins (e.g. Monsanto, Bayer, Syngenta). We will engage the public through generation of a website aimed at both a lay audience and at academic experts and we routinely promote of our findings through the general media (S4C, BBC), lay publications (usually to charities) and outreach activities aimed at school children. The agrochemical industry will be engaged through our existing links with Dow and Monsanto. Thus our research will provide major impact in several disparate areas.