When human and fungal worlds collide - a systems biology approach to understanding the dynamic interactions between human bronchial epithelial cells and conidiospores of Aspergillus fumigatus.

当人类和真菌世界发生碰撞时——一种系统生物学方法，用于了解人类支气管上皮细胞和烟曲霉分生孢子之间的动态相互作用。

• 批准号: RGPIN-2015-05043
• 负责人: Tebbutt, Scott
• 金额: $ 2.48万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=666634
• 关键词: When; human; fungal; worlds; collide

My research program will improve the scientific understanding, at the molecular and cellular levels, of how organisms from two disparate kingdoms (Animalia and Fungi) dynamically interact with one another, during co-culture. Specifically, I have selected human bronchial airway epithelial cells and spores from the fungus, Aspergillus fumigatus, due to the ubiquity of A. fumigatus whose spores are inhaled by all of us, each and every day. Furthermore, the genomes of both organisms have been sequenced and are well annotated, which will allow good biological inferences and hypothesis testing based on the gene expression (RNA) datasets generated through my shorter-term objectives (detailed below). Over the medium to longer term, I aim to extend my NSERC research program to incorporate proteomic and metabolomic studies, as well as direct experimental manipulations (eg, site-directed mutagenesis or siRNA knock-down) of genes that my current short-term objectives identify as important players in dynamic interactions between these spores and mammalian cells. Future refinement of the co-culture models will also be important, in order to explore additional cell types in the airway (eg, alveolar cells and dendritic cells).***My working hypothesis is that the interaction of A. fumigatus conidiospores with human primary bronchial epithelial cells leads to dynamic changes in the molecular profiles (transcriptome, proteome, metabolome) of both the fungal and human organisms.****Short-term objectives over the next 5 years of grant funding:***1. Develop an interaction model of A. fumigatus conidia with human primary bronchial epithelial cells, and measure spore uptake, as well as spore and cell death, over time using a variety of methods including fluorescence microscopy.***2. Use both unbiased and targeted molecular profiling technology (eg, RNA-Seq, nanoString nCounterr, SmartFlareT) to simultaneously measure dual organism gene expression changes in primary bronchial epithelial cells and A. fumigatus spores, during co-culture interaction.***3. Further develop and test a novel dual organism microfluidic-based device that will allow single human airway cells to come into contact with and interact with single A. fumigatus conidia.***The significance of the work outlined in this research program is several-fold: it will bring a new and unbiased transcriptomic perspective to the investigation of the interaction of A. fumigatus conidiospores with bronchial epithelial cells; application of nascent technologies including single cell/SmartFlare analysis and microfluidics will be applicable to other areas of research related to how organisms from different species interact at the cellular and molecular level; and multidisciplinary training of graduate and undergraduate students in areas as diverse as engineering, molecular biology, bioinformatics and biostatistics, and fungal and human cell biology.**

我的研究计划将在分子和细胞水平上提高对来自两个不同王国(动物界和真菌界)的生物体在共培养过程中如何动态相互作用的科学理解。具体地说，我选择了人类呼吸道上皮细胞和来自真菌烟曲霉菌的孢子，因为烟曲霉菌无处不在，我们每天都会吸入它的孢子。此外，这两种生物的基因组都已测序并作了很好的注释，这将使良好的生物学推断和假设检验基于我的短期目标(详述如下)生成的基因表达(RNA)数据集。从中长期来看，我的目标是扩展我的NSERC研究计划，包括蛋白质组和代谢组学研究，以及对基因的直接实验操作(例如，定点突变或siRNA敲除)，我目前的短期目标确定这些基因在这些孢子和哺乳动物细胞之间的动态相互作用中发挥重要作用。为了探索呼吸道中更多的细胞类型(如肺泡细胞和树突状细胞)，未来的共培养模型的改进也将是重要的。*我的工作假设是，烟曲霉分生孢子与人类原代支气管上皮细胞的相互作用导致真菌和人类生物体的分子图谱(转录组、蛋白质组、代谢组)的动态变化。*未来5年赠款资助的短期目标：*1.建立烟曲霉孢子与人原代支气管上皮细胞的相互作用模型，并测量孢子摄取、孢子和细胞死亡。使用包括荧光显微镜在内的各种方法随时间推移。*2.使用无偏向和靶向的分子图谱技术(例如，RNA-Seq、NanoStringnCounterr、SmartFlareT)同时测量原代支气管上皮细胞和烟曲霉孢子中双重生物基因表达的变化，在共培养过程中相互作用。*3.进一步开发和测试一种新的基于双生物微流控技术的装置，该装置将允许单个人呼吸道细胞与单个烟曲霉分生孢子接触和相互作用。*本研究计划中概述的工作的意义是多方面的：它将为研究烟曲霉分生孢子与支气管上皮细胞的相互作用带来一个新的、公正的转录学视角；包括单细胞/SmartFlare分析和微流控在内的新兴技术的应用将适用于与来自不同物种的生物如何在细胞和分子水平上相互作用有关的其他领域的研究，以及在工程学、分子生物学、生物信息学和生物统计学以及真菌和人类细胞生物学等领域对研究生和本科生进行的多学科培训。