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，人类主要真菌病原体的基因组序列得到了很好的注释，这为通过应用系统的全基因组方法彻底改变医学真菌学创造了机会。正如全基因组敲除集合的可用性有助于剖析酿酒酵母和pombe Schizosaccharomyces等模式真菌的生物学一样，在致病真菌中产生类似的完全基因缺失集合将创造实验工具，以系统地阐明真菌在哺乳动物宿主中的毒力基础，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菌，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，全世界三分之一的艾滋病患者死亡是由它造成的。利用优化的粒子轰击方法进行基因靶向，我们的实验室证明了在这种生物体中产生大量条形码基因缺失的可行性。具体来说，我们之前报道了1201株基因缺失菌株的构建。这个集合已经不受限制地提供给社区使用。我们通过系统筛选实验小鼠的传染性、已知毒力因子的表达、缺氧适应机制和吞噬抑制机制，证明了该集合的实用性。然而，这一收集的影响仅限于覆盖的基因部分——预测基因的五分之一。目的：我们建议生成和分发一个完整的新型隐球菌敲除突变体集合。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文库的定量筛选获得一系列参考表型，以了解体内感染性缺陷和体外主要毒力因子的表达。这项提议的工作将与其他旨在确定新生C.的基本基因的工作相协调。影响：提议的工作将改变医学真菌学，通过提供感染过程中适应性所需的病原体基因的第一个完整描述，对已知毒力因子生产所需的基因的完整描述，以及永久免费可用的，非冗余的零突变菌株资源，这将极大地加速整个领域的研究。

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.