Development of an in vitro model to dissect the mechanism of fungal persistence in the cystic fibrosis lung

囊性纤维化（CF）是高加索人群中最常见的致命性遗传疾病。这种疾病是由囊性纤维化跨膜调节基因（CFTR）突变引起的，该基因在上皮细胞中产生缺陷的离子通量和钙稳态。囊性纤维化患者易受真菌病原烟曲霉引起的肺部感染。动物模型在囊性纤维化的研究中起着核心作用。研究人员已经用老鼠、雪貂、猪和羊来模拟这种疾病的人类患者的炎症反应。虽然这些模型产生了关于这种疾病的病理生理学的有用知识，但由于一些伦理原因，动物模型受到限制。本研究拟采用CRISPR/Cas9诱变技术，建立一种新的体外等基因CF和健康对照支气管上皮细胞系携带不同CFTR基因突变的模型。使用我们实验室建立的方法学方法，包括体外感染、共聚焦显微镜、表达分析、细胞因子测量和竞争适应度分析，学生将能够定义囊性纤维化上皮和烟曲霉之间相互作用的关键阶段，与健康细胞和控制这一过程的真菌因素相比。这个项目的成功完成将实现若干科学目标。首先，上皮细胞系的等基因细胞培养系统的开发将对专注于CF上皮-病原体相互作用研究的研究人员具有重要价值。其次，CF上皮上烟曲霉零文库的筛选将增加我们对导致疾病持续存在的病原体因素的了解。此外，学生将参加一个课程，该课程将使学生有机会与曼彻斯特的工业合作伙伴和我们的真菌社区一起注册，以促进该资源在国际水平上的其他实验室的实施。

Cystic fibrosis (CF) is the most common fatal genetically inherited disease in Caucasian populations. This disease is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR) which produces defective ion fluxes and calcium homeostasis in the epithelia. Patients with cystic fibrosis are susceptible to lung infections caused by the fungal pathogen Aspergillus fumigatus.Animal models play a central role in the study of cystic fibrosis. Researchers have used mice, ferrets, pigs and sheep to model the inflammatory response seen in human patients with this disease. Although such models generate useful knowledge on the pathophysiology of this disease, animal models are limited for a number of ethical reasons.Here we propose to establish a new in vitro model of isogenic CF and healthy controls bronchial epithelial cell lines carrying different mutations in the CFTR gene using CRISPR/Cas9 mutagenesis. Using an established methodological approach in our laboratory including in vitro infection, confocal microscopy, expression analysis, cytokine measurements and competitive fitness analysis the student will be able to define critical stages of the interaction between the cystic fibrosis epithelia and A. fumigatus compared to healthy cells and the fungal factors governing this process.The successful completion of this project will achieve a number of scientific objectives. Firstly, the development of an isogenic cell culture system of epithelial cell lines will be of great value for researchers focused on the study of epithelia-pathogen interactions in CF. Secondly, screening of A. fumigatus null libraries on CF epithelia will increase our knowledge on the pathogen factors leading persistence in the context of disease. Additionally, the student will be enrolled in a programme that will give the student the opportunity to enroll with industrial partners in Manchester and our fungal community to promote the implementation on this resource in other laboratories at an international level.