Establishment of the thermotolerant Arabian killifish as a model for infection studies

近年来，斑马鱼被广泛用于研究免疫系统与一系列导致人类疾病的微生物之间的相互作用，但这些研究都没有反映温度对毒力性状表达的重要性。因此，我们特别选择了阿拉伯鳉鱼（Aphanius dispar）作为这项提议的对象，因为它自然地适应高温环境（37-40摄氏度），这使我们能够研究微生物感染的病理生物学，以及病原体在与我们自身温度一致的情况下的行为。该提案建立在我们已经在一个博士项目（Kudoh, Ramsdale & Hamied）中开展的工作基础上，该项目旨在开发真菌（白色念珠菌）感染模型。到目前为止，我们的模型已经显示出斑马鱼模型的所有优点，包括出色的活细胞荧光和明场成像，抗真菌筛选，以及（正如我们所展示的）创建转基因和CRISPR/Cas9突变系的能力。在我们提出的项目中，我们现在有以下科学目标：(1)产生转基因鱼系，促进关键免疫细胞群的活细胞成像-取代啮齿动物模型。(2)产生具有与人类疾病相关的基因缺陷的突变鱼种，这些基因缺陷是感染的诱发因素——取代啮齿类动物模型进行机械感染研究。(3)基于我们现有的序列数据，建立一个公开可用的、可搜索的阿拉伯鳉鱼基因组数据库/门户网站，使社区能够在我们的系统中设计实验。利用来自不同胚胎发育阶段和不同温度（30℃和37℃）感染的RNAseq数据，绘制出免疫组的关键标记，这些标记可以作为鳉鱼基因组查看器上的轨迹，以便模型的用户可以评估特定阶段的病理生物学相关事件。(4)向更广泛的感染生物学研究界传播我们的工具（鱼线）、数据（免疫组）和方法（成像和感染），并为斑马鱼、小鼠和其他模型的研究人员提供机会，将阿拉伯鳉鱼作为替代模型。与啮齿动物和斑马鱼相比，我们的NC3Rs模型具有以下优势：替代（部分）：我们的阿拉伯鳉鱼胚胎模型可以替代许多用于研究与免疫细胞和组织相互作用、初始毒力测试和抗真菌/抗菌药物筛选的小鼠。在埃克塞特大学医学真菌学MRC中心5年的研究中，当地设施的总剂量为25% = 1,700只小鼠。减少：在阿拉伯，可以从单个活动物感染的不同阶段收集鳉鱼数据，大大减少了不必要的扑杀老鼠和相关的高饲养成本。假设五年内的使用率为5%，那么用于感染研究的45687只小鼠就可以减少。改进：斑马鱼胚胎中人类病原体的标准筛选在32-33℃进行，施加不必要的热应力。从科学上讲，该模型具有更大的有效性，因为人类病原体需要在37摄氏度的温度下生长，以表达其全套毒力特征。由于发育性抽搐减少，对三卡因麻醉的需求也减少了。每年总共有74820只斑马鱼被使用（基于文献分析），所以在5年的时间里，如果有5%的比例，就可以对18750只斑马鱼的胚胎进行研究。作为埃克塞特大学MRC医学真菌学中心（CMM - Ramsdale和Farrer）和水生资源中心（ARC - Kudoh）的核心成员，我们在鱼类生物学、真菌分子和细胞生物学、细胞生物学、免疫学和生物信息学方面拥有最先进的专业知识，以支持这项工作。因此，埃克塞特提供了一个充满活力，令人兴奋和科学优秀的研究环境，是执行这些研究的完美场所。

Zebrafish have been widely used in recent years to study the interaction of the immune system with a range of microbes that cause human disease - yet none of these reflects the importance of temperature on the expression of virulence traits. We have therefore specifically chosen the Arabian killifish (Aphanius dispar) for this proposal as it is naturally acclimated to high temperature environments (37-40'C) - allowing us to investigate the pathobiology of microbial infections, and the behaviour of pathogens at temperatures consistent with our own. The proposal builds upon work we have already undertaken in a doctoral project (Kudoh, Ramsdale & Hamied) to develop the model for fungal (Candida albicans) infections. So far our model has shown all of the benefits of the zebrafish model, including excellent live cell fluorescence and brightfield imaging, antifungal screens, and (as we have shown) the ability to create transgenic and CRISPR/Cas9 mutant lines. In the project we are proposing, we now have the following scientific aims:(1) Generate transgenic fish lines that facilitate live cell imaging of key immune cell populations - replacing rodent models.(2) Generate mutant fish lines with defects in genes linked to human disease that are predisposing factors for infection - replace rodent models for mechanistic infection studies. (3) Establish a publicly available, searchable Arabian killifish genome database /portal based on our existing sequence data - to allow the community to design experiments in our system. Use RNAseq data from different stages of embryo development and infection at different temperatures (30'C and 37'C) to map out key markers of the immunome that can be made available as a track on the killifish genome viewer so that users of the model can assess stage specific pathobiology related events.(4) Disseminate our tools e.g. (fish lines), data (immunome) and methods (imaging and infection) to the wider infection biology research community and provide opportunities to researchers in zebrafish, mice and other models to use the Arabian killifish as an alternative model. In relation to NC3Rs our model has the following advantages over rodents and zebrafish:Replacement (Partial): Our Arabian killifish embryo model could replace many mice that are used for studies of interactions with immune cells and tissues, initial virulence testing and antifungal / antibacterial drug screens. TOTAL in local facilities, with 25% uptake within the MRC Centre for Medical Mycology at the University of Exeter over 5 years = 1,700 mice.Reduction: In the Arabian killifish data can be collected from different stages of infection in a single live animal, greatly reducing the unnecessary culling of mice and associated high husbandry costs. Assuming 5% take-up over five years a reduction in use of 45,687 mice for infection studies could be achieved.Refinement: Standard screens of human pathogens in zebrafish embryos are undertaken at 32-33'C , imposing unnecessary thermal stress. Scientifically the model has greater validity as human pathogens need to grow at 37'C in order to express their full suite of virulence traits. There is also a reduced need for tricaine anaesthesia due to less developmental twitching. A TOTAL of 74,820 zebrafish are used p.a (based on literature analysis) so with a 5% take up over five years 18,750 zebrafish embryo studies could be refined.As core members of the MRC Centre for Medical Mycology (CMM - Ramsdale and Farrer) and the Aquatic Resource Centre (ARC - Kudoh) at the University of Exeter we have state-of-the-art expertise in fish biology, fungal molecular and cell biology and cell biology, immunology and bioinformatics to support this work. Exeter therefore provides a vibrant, exciting and scientifically excellent research environment, and is the perfect place for these studies to be executed.