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

被忽视的热带病血吸虫病影响低收入和中等收入国家的2.5亿多人，其中6亿多人面临风险。该病由血吸虫属的血吸虫感染引起，根据种类不同，与肝脏炎症和纤维化或膀胱和肾脏病理有关。迫切需要新的控制策略来打破对单一药物（吡喹酮）的完全依赖。此外，耐药性的威胁正在出现。与大多数雌雄同体的扁虫不同，血吸虫有单独的雄性和雌性个体。研究的当务之急是了解血吸虫不寻常的发育和性生物学，因为阻断传播的策略可能会被揭示。虽然寄主因素可能参与了性分化，但通过体外转化幼虫获得的培养寄生虫可以发育成性分化的蠕虫，尽管与体内发育相比延迟并且不能产生活卵。因此，一种内在的固有程序构成了性别分化的基础；然而，在哺乳动物宿主内，单型幼虫分化为具有不同性别形式的生物体所涉及的分子参与者仍未被广泛探索。“大量”转录组学研究表明，在两性形态差异出现之前，两性转录组二态性在发育早期就已经很好地建立起来了。然而，这些对整个蠕虫的研究缺乏识别低表达或在少数细胞中表达的性别偏倚基因的分辨率，并且由于来自不同细胞群体的信号聚集，可能产生误导性的结果。此外，在血吸虫中鉴定出性别偏倚基因表达的研究并没有从功能上描述性别偏倚基因。该项目的总体目标是确定和表征在早期哺乳动物阶段曼氏s.m ansoni两性二态性建立中涉及的关键基因和途径。提出了两个互补但独立的工作包。在WP-I中，我们将使用单细胞RNA-seq （scRNA-seq）来准确识别与感染和与哺乳动物宿主早期相互作用相关的发育阶段（包括研究较少的肺阶段）性别差异表达的基因和途径。性别偏向的细胞群，他们的发展轨迹随时间将被描述。优先基因将通过基因沉默和寄生虫表型在男性和女性中进行功能评估。对于与稳健表型相关的基因，第二个更细粒度的表型将紧随其后；敲除的寄生虫将被解离并通过scRNA-seq进行分析。scRNA-seq和基因沉默都是我和其他人在血吸虫中开发的成熟方法。WP-II的重点是技术开发，其中将研究精制培养系统，以改进血吸虫发育的体外研究，包括寄生虫性成熟。其次，我们将优化产生突变寄生虫的替代方法，这建立在我将CRISPR-Cas应用于血吸虫的成功基础上。这将提供一个应急计划来扰乱基因，特别是那些对RNAi难解的基因。总体结果将导致更好地理解早期哺乳动物内部发育和血吸虫的基本性生物学。我们将建立大规模开放获取的“细胞图谱”数据库，并为血吸虫和其他蠕虫的功能工具箱做出贡献。阿伯里斯特威斯大学生物、环境与农村科学研究所拥有寄生虫材料、表型平台和基本的寄生虫学经验，是开展这项研究最合适的地方。此外，Wellcome Sanger研究所和UT Southwestern （USA）的合作者将分别提供生物信息学支持和功能基因组学专业知识。

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.