Structural biology of immune receptors in disease-carrying mosquitoes Mosquitoes are among the deadliest insects as they are reservoirs of pathogenic organisms that cause life-threatening diseases including malaria, yellow and dengue fever among others. These diseases are transmitted from host to host by the mosquito during their blood meal. It has been reported that an infectious mosquito has an increased appetite, which boosts the transmission efficiency of the germs.  It is currently not clear how the mosquito deals with the infection and how the germs bypass the mosquito's immune system. What is known so far comes mostly from the study of the fruit fly Drosophila melanogaster that serves as a model organism for insects. Considerable insight has been gained from this system and led to the discovery of key signalling pathways in innate immunity conserved throughout the animal kingdom. The TOLL pathway is of particular interest to me as it combines functions during development and adult life, in the immune and nervous systems. This makes the TOLL pathway a multifaceted target for intervention. My main objective is to characterize the mechanism of TOLL activation and signalling in mosquitoes.   Members of the TOLL family are membrane proteins with an extracellular region responsible for ligand recognition, a single-spanning transmembrane region and an intracellular domain able to transmit the signal inside the cell. The cell, in turn, produces antimicrobial peptides and coordinates different cellular processes in order to mount the appropriate immune reaction against the infection. My first aim is to characterize the three-dimensional structures of mosquito TOLL receptors providing size, shape and charge information. The second aim is to characterize the complex formed with their ligand to reveal their mechanism of activation.  Both aims will be tackled using computational approaches of protein modelling, based on their common ancestry with Drosophila TOLL1, whose structure is known in the absence and presence of its ligand Spatzle 1 (SPZ1). SPZ1 is a signalling molecule that belongs to the same family of proteins as mammalians neurotrophins (NTs), which have recently been involved in physiological mechanisms regulating food intake, as well as vertebrate innate immunity. The recognition mechanism of Drosophila SPZ by TOLL is governed by shape and electrostatic complementary, as well as spatial restriction due to "sugar-coating" at the surface of the receptor. Predictive docking of mosquito complexes relies on coevolution at protein interfaces. These predictions will be verified experimentally using biophysical techniques, X-ray crystallography and cryo-electron microscopy for atomic structure determination and cell-based functional studies, such as mutagenesis, reporter gene assays and fluorescence microscopy. Cellular signalling assays will be based on transiently transfected cells stimulated by SPZ and NT isoforms or microbial signature molecules, as appropriate, in order to trigger cellular responses in the form of bioluminescence. In turn fluorescence microscopy will localise fluorescence-tagged molecules inside the cells and colocalise two differently-labeled molecules.      While there is a lot of data on fruit fly TOLL1 and human TOLL-like receptors, there is currently no structural information on mosquito TOLL receptors and SPZ/NT ligands, which have diverged and undergone gene duplication under the influence of parasitic pressure, possibly to acquire new functions. I expect therefore to identify potential novel mechanisms of ligand binding and signalling.  This information will be useful to understand the mechanism of TOLL signalling in mosquitoes in general and establish a link between immune and nervous systems in particular, which might play a dual role in controlling the physiology and the behaviour of infectious mosquitoes. Harnessing information on TOLL-mediated crosstalk has the potential to guide the development of new strategies for pest control. The Global Challenges Research Fund (GCRF) supports cutting-edge research to address challenges faced by developing countries. The fund addresses the UN sustainable development goals. It aims to maximise the impact of research and innovation to improve lives and opportunity in the developing world.