Functional genomics to unveil the early intra-mammalian development of schistosomes

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The Neglected Tropical Disease schistosomiasis affects more than 250 million people in Low and Middle-Income Countries, where more than 600 million are at risk. The disease, caused by infection with blood flukes in the genus Schistosoma, is associated with liver inflammation and fibrosis, or bladder and kidney pathology, depending on the species. New control strategies are urgently needed to break the complete reliance on a single drug (praziquantel). Moreover, the threat of drug resistance is emerging. Unlike most hermaphroditic flatworms, schistosomes have separate male and female individuals. A research imperative is to understand the unusual development and sexual biology of schistosomes because strategies to block transmission may be revealed. Although host factors might be involved in the sexual differentiation, cultured parasites obtained by in vitro transformation of larvae can develop into sexually differentiated worms, albeit delayed compared to in vivo development and incapable of producing viable eggs. Thus, an intrinsic hardwired program underlies sexual differentiation; however, the molecular players involved in the differentiation of monomorphic larvae into organisms with distinct sexual forms within the mammalian host remain largely unexplored. "Bulk" transcriptomic studies suggest that a sexual transcriptomic dimorphism is well established early in development long before morphological differences between the sexes appear. However, these studies on whole worms lack the resolution to identify sex-biased genes with low expression or expressed in few cells, and can generate misleading results due to signal aggregation from different cell populations. Moreover, studies that identified sex-biased gene expression in schistosomes did not functionally characterise sex-biased genes. The overarching goal of this project is to identify and characterise key genes and pathways involved in the establishment of sexual dimorphism in early intra-mammalian stages of S. mansoni. Two complementary but independent work packages (WP) are proposed. In WP-I, we will use single cell RNA-seq (scRNA-seq) to accurately identify genes and pathways differentially expressed between sexes in developmental stages associated with infection and early interactions with the mammalian host, including the poorly-studied lung stage. Sex-biased cell populations, and their developmental trajectories over time will be characterised. Prioritised genes will be functionally assessed in males and females by gene silencing and parasite phenotyping. For genes associated with robust phenotype(s), a second finer-grained phenotyping will follow; the knocked-down parasite will be dissociated and analysed by scRNA-seq. Both scRNA-seq and gene silencing are well-established approaches that I and others have developed in schistosomes. WP-II focuses on technology development where refined culture systems will be investigated to improve the in vitro study of schistosome development, including parasite sexual maturation. Second, we will optimise alternative approaches to generate mutant parasites, which builds on my successes in applying CRISPR-Cas to schistosomes. This will provide a contingency plan to perturb genes, in particular those refractory to RNAi.The overall outcome will lead to a better understanding of the early intra-mammalian development and basic sexual biology of schistosomes. We will generate large-scale open access 'cell-atlas' databases, and contribute to the functional toolbox for schistosomes and other helminths. Access to parasite material, phenotyping platforms, and basic helminthology experience positions the Institute of Biological, Environmental & Rural Sciences, Aberystwyth University, the most suitable place to undertake this research. In addition, collaborators at the Wellcome Sanger Institute and UT Southwestern (USA) will contribute with bioinformatic support and functional genomics expertise, respectively.