Cryptococcus neoformans Gene Knockout Resource

描述(申请人提供)：有效诊断和治疗人类危及生命的真菌感染是一项尚未满足的重大临床挑战，医学真菌学是解决这一需求的研究学科。最近发表了注释良好的主要人类真菌病原体的基因组序列，创造了通过应用系统的全基因组方法来革命医学真菌学的机会。正如全基因组敲除集合的可获得性有助于剖析酿酒酵母和裂殖酵母等模式真菌的生物学一样，病原真菌中类似的完整基因缺失集合的产生将创造出实验工具，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。新生隐球菌是一种被包裹的芽生酵母，是引起真菌性脑膜炎的最常见原因。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于艾滋病。使用优化的基因打靶粒子轰击方法，我们的实验室证明了在这种生物中产生大量条形码基因缺失的可行性。具体地说，我们之前报道了1201个基因缺失菌株的构建。这些收藏品已向社区提供，不受限制。我们通过对实验小鼠的感染性、已知毒力因子的表达、低氧适应机制和吞噬抑制机制的系统筛选，证明了该收集的实用性。然而，这种收集的影响仅限于所覆盖的基因的一部分--预测基因的五分之一。目的：我们建议产生并分发一套完整的新生隐球菌基因敲除突变株。基因敲除将包含有助于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中体内感染性缺陷和主要毒力因子在体外的表达来获得一系列参考表型。建议的efort将与其他旨在识别新生葡萄球菌基本基因的研究相协调。影响：拟议的工作将改变医学真菌学，通过提供第一个全面描述感染期间适应所需的病原体基因，完整计算产生已知毒力因子所需的基因，以及一个永久的免费可用、非冗余的零突变菌株资源，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.