Chemical-genetic functional annotation of the genome of a meningitis pathogen

描述(由申请人提供)：理论基础：有效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。人类主要真菌病原体的注释基因组序列现在可用，创造了通过系统方法的应用来革命医学真菌学的机会。特别是，由于模式酵母中的全基因组敲除收集已经有助于剖析真核细胞的基本过程，病原真菌中类似基因缺失收集的产生和分析现在开始允许系统地阐明哺乳动物宿主中毒力的分子决定因素。新生隐球菌是人类最重要的三种真菌病原体之一。它在实验上是非常容易驯服的，拥有一个完整的性周期，服从于遗传学和出色的感染动物模型。这种机会主义的微囊化发芽酵母是引起真菌性脑膜炎的最常见原因。据估计，每年有100万个病例导致约60万人死亡，三分之一的艾滋病患者的死亡归因于这种病原体。利用优化的基因打靶方法，我们实验室构建了1201株基因缺失菌株文库。我们利用这一资源对实验小鼠的病原体适应性、已知毒力因子的表达、低氧适应机制和吞噬抑制机制进行了系统的筛选。然而，在这些筛查中发现的许多基因，虽然对致病性至关重要，但具有未知的分子功能。因此，对毒力的进一步研究需要一些方法，这些方法可以导致对新基因的功能注释。目的：我们提出应用化学发生谱这一强大的化学遗传学方法来解决功能注释的问题。在这种方法中，化学扰动对大量定义的基因敲除的适合度的影响被量化。由此产生的表型指纹被用来将基因聚集成功能基团，并定义它们彼此之间的角色。我们建议将这种方法应用于新生葡萄球菌，从功能上注释致病所必需的关键基因。为了实现这一目标，我们将首先开发一个项目信息学基础，并确定适合于化学遗传学分析的生物活性化学物质。然后，我们将获得并分析对一系列生物活性化学物质的全稀释定量适应性反应。最后，我们将利用这些结果来开发和测试具体的假设，以确定未知功能的基因产物在宿主中的病原体适合性中所起的作用。影响：拟议中的工作将产生第一份详细的人类微生物病原体基因组大部分的表型图。通过将具有相似图谱的基因聚集在一起，这些研究有望导致对先前哺乳动物感染研究中确定的致病因子的分子功能的迫切需要的深入了解。此外，这项工作有望定义基本毒力途径的化学调节器，有望成为病原体生物学研究和未来治疗开发的有力工具。

DESCRIPTION (provided by applicant): Rationale: The effective treatment of life-threatening fungal infections in humans is a major unmet clinical challenge. Annotated genomic sequences of the major human fungal pathogens are now available, creating an opportunity to revolutionize medical mycology through the application of systematic approaches. In particular, as genome-wide knockout collections in model yeasts have been instrumental to the dissection of fundamental eukaryotic cellular processes, the generation and analysis of analogous gene deletion collections in pathogenic fungi is now beginning to allow the systematic elucidation of the molecular determinants of virulence in the mammalian host. Cryptococcus neoformans is one of the three most important human fungal pathogens in humans. It is highly tractable experimentally, having a complete sexual cycle amenable to genetics and outstanding animal models for infection. This opportunistic encapsulated budding yeast is the most common cause of fungal meningitis. Annually, there are estimated 1,000,000 cases that result in ~600,000 deaths, and one-third of deaths in AIDS patients are attributed to this one pathogen. Using optimized methods for gene targeting, our laboratory constructed a library 1201 gene deletion strains. We exploited this resource for systematic screens of pathogen fitness in experimental mice, expression of known virulence factors, mechanisms of hypoxic adaptation, and mechanisms of phagocytosis-inhibition. However, many of the genes identified in these screens, while critical for pathogenicity, are of unknown molecular function. Thus, obtaining further insigh into virulence requires methods that can lead to the functional annotation of novel genes. Objective: We propose to address the problem of functional annotation by applying chemogenetic profiling, a powerful chemical-genetic method. In this approach, the impact of chemical perturbations on the fitness of large numbers of defined gene knockouts is quantified. The resulting phenotypic fingerprints are used to cluster genes into functional groups and to define their roles with respect to each other. We propose to apply this method to C. neoformans to functionally annotate key genes necessary for pathogenicity. To accomplish this goal, we will first develop a project informatics foundation and to identify bioactive chemicals suitable for chemogenetic profiling. We will then obtain and analyze full-dilution quantitative fitness responses to an array of bioactive chemicals. Finally, we will exploit the results to develop and test concrete hypotheses for the role of gene products of unknown function that we have previously implicated in pathogen fitness in the host. Impact: The proposed work will produce the first detailed phenotypic map of a large portion of the genome of any human microbial pathogen. By clustering genes with similar profiles together, these studies are anticipated to lead to critically-needed insight into the molecular functions of pathogen factors identified in ou previous studies of mammalian infection. In addition, this work is anticipated to define chemical modulators of essential virulence pathways, which are expected to be powerful tools for studies of pathogen biology and future therapeutic development.