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

描述（由申请方提供）：依据：有效治疗危及生命的人类真菌感染是一项重大的未满足的临床挑战。人类主要真菌病原体的注释基因组序列现已可用，这为通过应用系统方法彻底改变医学真菌学创造了机会。特别是，作为全基因组敲除收集在模型酵母已有助于解剖的基本真核细胞过程，类似的基因缺失收集在致病真菌的生成和分析，现在开始允许系统阐明的分子决定因素的毒力在哺乳动物宿主。新型隐球菌是人类三大重要的真菌病原体之一。它在实验上非常容易控制，有一个完整的性周期，适合遗传学和杰出的感染动物模型。这种机会性包囊芽殖酵母是真菌性脑膜炎最常见的原因。每年估计有1，000，000例病例，导致约600，000人死亡，三分之一的艾滋病患者死亡归因于这一病原体。本实验室采用优化的基因打靶方法，构建了1201株基因缺失菌株的文库。我们利用这一资源进行系统的筛选病原体健身实验小鼠，表达已知的毒力因子，缺氧适应机制，和吞噬抑制机制。然而，在这些筛选中鉴定的许多基因虽然对致病性至关重要，但其分子功能未知。因此，获得进一步深入到毒力需要的方法，可以导致新基因的功能注释。目的：我们建议通过应用化学遗传学分析，一种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应性的影响被量化。由此产生的表型指纹被用于将基因聚类为功能组，并定义它们彼此之间的作用。我们建议将这种方法应用于C。新形式的功能注释致病性所必需的关键基因。为了实现这一目标，我们将首先开发一个项目信息学基础，并确定适合化学遗传学分析的生物活性化学品。然后，我们将获得和分析一系列生物活性化学品的全稀释定量健身反应。最后，我们将利用这些结果来开发和测试我们以前在宿主中与病原体适应性有关的未知功能的基因产物的作用的具体假设。影响：拟议的工作将产生任何人类微生物病原体基因组大部分的第一个详细表型图。通过将具有相似特征的基因聚类在一起，这些研究预计将导致对在先前的哺乳动物感染研究中鉴定的病原体因子的分子功能的迫切需要的了解。此外，这项工作预计将确定基本毒力途径的化学调节剂，预计将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.