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.

This project combines my multifaceted technical skills applied to the characterization of the structural biology of mosquito immune receptors. I will focus on TOLL receptors implicated in resistance to pathogens that have undergone gene duplication in disease-carrying mosquitoes.

I will combine structural techniques (X-ray crystallography and electron microscopy) with biophysical tools and molecular modelling to understand the mechanism of activation of TOLL1 and 5 by SPATZLE1 (SPZ1) isoforms; TOLL7 by neurotrophin1 (NT1, also SPZ2) and viral envelope glycoproteins; and orphan TOLL9 and 11. I will use baculovirus expression to produce the extracellular ligand-binding domains of these receptors. Bacterial and insect expression systems are foreseen for the production of SPZ and NT isoforms, the respective TOLL ligands. Ligand fishing experiments with Biacore will help identify the ligands of orphan TOLL9 and 11.

I have successfully produced fruit fly TOLL1 and SPZ1 and determined the crystal structure of their 1:1 complex at a resolution of 2.4 Å. Here I will use mosquito TOLL5A and SPZ1C, with established immune functions, to determine their atomic structure. My working hypothesis is that using a different species will facilitate the crystallization of the active dimer in a 2:2 complex, which eluded me using Drosophila material. Comparing monomer and dimer structures will establish the molecular basis of negative cooperativity.

I will characterize NT1 and viral glycoprotein recognition by TOLL7 and compare to binding modes of TOLL, mammalian TLRs, and also NT receptors p75, trk and sortilin that have no direct homologue in mosquitoes.

Cellular signalling assays will confirm the validity of my structural observations. Fluorescence microscopy will determine the cellular localization of the activated receptors and co-localization of adaptors. Structure-function relationships analysis will give insight into the mechanism of protein network evolution.

This project aims at expanding our basic knowledge of how the mosquito's immune system recognizes pathogens and activates protective signaling pathways. Description at the atomic level of the interaction between TOLL and SPZ that have protective role against plasmodium and Dengue virus infection has tremendous impact for the development of new strategies to counteract the spread of multi-drug resistant parasites and the emergence of insecticide-resistant vectors. Based on SPZ's neurotophic activity, this project has the potential to tackle neuroimmunology-related questions that might apply throughout the animal kingdom and help advances in the characterization of the crosstalk between nervous and innate immune systems.

Results of the research will be published fully, and without delay. Dissemination of the results of the research outlined will be by publication in international open access journals of the highest standard. In the case of high profile results, the press office at Cambridge is ready to accompany the publication with a press release. Funds are requested for presentation at conferences that will be used to disseminate the results in a timely manner to the appropriate specialized audience (e.g. Innate Immunity, Structural Biology, Neuroimmunology).

This project will be a stepping-stone in my career to a position of independence and maturity while developing further transferable skills and exposure to the scientific and, potentially, the medical community and the industrial sector. I will reinforce communication skills by writing grant reports and scientific publications and communications. I will develop lasting cooperation and collaborations within the UK and with other countries to contribute to the excellence of the Research Organization

Staff training is an objective of the project, hence the request for a postdoctoral RA. If the approach I plan to develop is successful, the skills and capabilities achieved by this training program will increase the availability of a highly skilled worker in the UK that will be an advantage in a knowledge-based economy. The researcher trained in this project will be in a position to make a valuable and practical contribution to the continued growth of the biochemical and possibly the pharmaceutical sector in the UK.

My thinking outside the box is illustrated in a single-authored opinion article that has been cited 31 times by international researchers in the last four years (Trends Biochem Sci. 2012; 37(3): 92-98). For this project I plan the development of original signaling strategies to study the mechanism of activation of immune receptors in disease-carrying mosquitoes. It is possible that technical aspects of this project will have commercial appeal and require IP protection. I am familiar with the pathway to commercialization, as I have patented a novel expression system in the past. The proof of concept stage was funded by the technology transfer office (TTO) at Cambridge Enterprise. I had regular meetings with technology transfer officers to structure the process of commercialization; special attention was paid to coordinate IP protection and timely presentation at conferences. The TTO had arranged all steps for drafting and filing a national patent, which was presented to investors at technology fairs and conferences. International patent filing under the Patent Commercialization Treaty (PCT) did not go ahead due to market size.

This project is related to fighting human diseases as widely spread as malaria and haemorrhagic fever that are transmitted by mosquitoes and constitute a medical priority. In contrast to the study of fruit flies, the fame or rather the infamy of mosquitoes will facilitate media exposure and communication and engagement with the general public very aware of the problem affecting a large portion of the human population.