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.

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领域（动物和真菌）的生物在共培养过程中如何动态相互作用的科学理解。具体来说，我选择了人类支气管气道上皮细胞和真菌烟曲霉的孢子，因为烟曲霉无处不在，我们每个人每天都吸入它的孢子。此外，这两种生物的基因组已经测序并进行了很好的注释，这将允许基于我的短期目标（详细如下）生成的基因表达（RNA）数据集进行良好的生物学推断和假设检验。在中长期内，我的目标是扩展我的NSERC研究项目，纳入蛋白质组学和代谢组学研究，以及我目前的短期目标确定为这些孢子和哺乳动物细胞之间动态相互作用的重要参与者的基因的直接实验操作（例如，位点定向诱变或siRNA敲除）。为了探索气道中的其他细胞类型（如肺泡细胞和树突状细胞），未来对共培养模型的改进也将是重要的。

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 nCounter®, SmartFlare™) 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.