Cryptococcus neoformans Gene Knockout Resource

描述（由申请人提供）：有效诊断和治疗危及生命的人类真菌感染是一个主要的未满足的临床挑战，医学真菌学是解决这一需求的研究学科。最近发表了人类主要真菌病原体的注释良好的基因组序列，通过系统的全基因组方法的应用，创造了一个革命性的医学真菌学的机会。正如全基因组敲除集合的可用性已经有助于解剖模式真菌的生物学，如酿酒酵母和粟酒裂殖酵母，在致病性真菌中产生类似的完整基因缺失集合将创建实验工具，以系统地阐明哺乳动物宿主中真菌毒力的基础，并开发针对它们的药物。 新型隐球菌是一种被包裹的芽殖酵母，是真菌性脑膜炎最常见的原因。据估计，每年有100万例病例导致约60万人死亡。据估计，它造成了全世界艾滋病患者死亡的三分之一。使用优化的基因靶向粒子轰击方法，我们的实验室证明了在这种生物体中创建大量条形码基因缺失的可行性。具体而言，我们以前报道了1201基因缺失菌株的构建。这些收藏品已不受限制地向社区提供。我们证明了这个集合的效用，通过使用它在实验小鼠的感染性，已知的毒力因子的表达，缺氧适应机制，和吞噬抑制机制的系统屏幕。然而，这种收集的影响仅限于所覆盖的基因的比例-预测基因的五分之一。目的：我们建议产生和分发一个完整的收集敲除突变的新生隐球菌。敲除将包含促进功能基因组研究的特征。我们进一步建议通过定量筛选文库的体内感染性缺陷和体外主要毒力因子的表达来获得一系列参考表型。建议的efort将协调与其他人的目标是确定C的必需基因。新人类影响力：拟议的工作将通过提供感染期间健身所需的病原体基因的第一个完整描述，已知毒力因子生产所需的基因的完整说明，以及永久免费，非冗余的无效突变菌株资源，将大大加速整个领域的研究来改变医学真菌学。

DESCRIPTION (provided by applicant): The effective diagnosis and treatment of life-threatening fungal infections in humans is a major unmet clinical challenge, and medical mycology is the research discipline that addresses this need. Well-annotated genomic sequences of the major human fungal pathogens have recently been published, creating an opportunity to revolutionize medical mycology through the application of systematic genome-wide approaches. Just as the availability of genome-wide knockout collections has been instrumental in dissecting the biology of model fungi such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, the generation of analogous complete gene deletion collections in pathogenic fungi would create experimental tools to systematically elucidate the basis of fungal virulence in the mammalian host and to develop drugs against them. Cryptococcus neoformans is an encapsulated budding yeast that is the most common cause of fungal meningitis. There are an estimated 1,000,000 cases that result in ~600,000 deaths each year. It is estimated to cause one-third of deaths in AIDS patients worldwide. Using optimized particle bombardment methods for gene targeting, our laboratory demonstrated the feasibility of creating large numbers of bar-coded gene deletions in this organism. Specifically, we reported previously the construction of 1201 gene deletion strains. This collection has been made available without restriction to the community. We demonstrated the utility of this collection by using it in systematic screens of infectivity in experimental mice, expression of known virulence factors, mechanisms of hypoxic adaptation, and mechanisms of phagocytosis-inhibition. However, the impact of this collection is limited to the fraction of genes covered -- one-fifth of predicted gens. Objectives: We propose to generate and distribute a complete collection of knockout mutants for Cryptococcus neoformans. Knockouts will contain features to facilitate functional genomic studies. We further propose to obtain a series of reference phenotypes via quantitative screens of the library for defects in infectivity in vivo and the expression of the major virulence factorsin vitro. The proposed efort will be coordinated with those of others that aim to identify the essential genes of C. neoformans. Impact: The proposed work will transform medical mycology by providing the first full description of pathogen genes required for fitness during infection, a ull accounting of genes required for production of known virulence factors, and a permanent freely-available, nonredundant null mutant strain resource that will dramatically accelerate research in the field as a whole.