Investigation of uncharacterized fungal-specific genes in dormant conidia

项目摘要 许多侵袭性真菌疾病始于从环境中吸入的孢子打破休眠。 并在宿主体内开始活跃的生长。尽管它们对真菌的生命周期至关重要 和潜在的抗真菌靶标，孢子休眠的建立和维持是 人们对此知之甚少。 拟议的对人类病原体烟曲霉的初步研究侧重于真菌- 在休眠的无性孢子中具有高转录水平的未知功能的特定基因 (分生孢子)和生长活跃的细胞中的低水平。具有这种特殊表达模式的基因， 指定的hic(高分生孢子)基因，很可能参与建立或 孢子休眠的维持。在拟议的先导研究中，25个hic基因中 休眠和非休眠细胞中的差异转录水平将通过 删除和标记。将分析缺失菌株的发育、休眠、 用流式细胞仪和显微镜观察种子萌发情况。将对标记的菌株进行分析 Hic蛋白在发育、休眠和萌发过程中的荧光定位 显微镜。标记的菌株还将用于亲和纯化和质谱分析 识别相互作用的蛋白质。 拟议的初步研究的成功完成将确定一组对 真菌分生孢子休眠的建立和维持及其定位和相互作用 发育过程中进入休眠状态以及从休眠状态进入活跃生长状态的数据。

Project Summary Many invasive fungal diseases start when spores inhaled from the environment break dormancy and begin active growth within the host. Despite their critical importance to the fungal life cycle and potential as antifungal targets, the establishment and maintenance of spore dormancy are poorly understood. The proposed pilot study in the human pathogen Aspergillus fumigatus focuses on fungal- specific genes of unknown function that have high transcript levels in dormant asexual spores (conidia) and low levels in actively growing cells. Genes with this particular expression pattern, designated hic (high in conidia) genes, are likely to be involved in the establishment or maintenance of spore dormancy. In the proposed pilot study, the 25 hic genes with the most differential transcript levels in dormant vs nondormant cells will be characterized through deletion and tagging. Deletion strains will be analyzed for changes in development, dormancy, and germination using flow cytometry and microscopy. Tagged strains will be analyzed for localization of Hic proteins during development, dormancy, and germination using fluorescence microscopy. Tagged strains will also be used in affinity purification and mass spectroscopy to identify interacting proteins. Successful completion of the proposed pilot study will identify a set of genes important for establishing and maintaining dormancy in fungal conidia along with localization and interaction data for the transition to dormancy during development and from dormancy to active growth.