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

描述（由申请人提供）：理由：对危及生命的真菌感染的有效治疗是一个主要的未满足的临床挑战。主要人类真菌病原体的注释基因组序列现在是可用的，创造了一个机会，通过应用系统的方法来彻底改变医学真菌学。特别是，由于模型酵母的全基因组敲除集合有助于解剖真核细胞的基本过程，致病真菌中类似基因缺失集合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。新型隐球菌是三种最重要的人类真菌病原体之一。它在实验上是高度可控的，有一个完整的性周期，符合遗传学和优秀的感染动物模型。这种机会性包封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100万例艾滋病病例导致60万例死亡，其中三分之一的艾滋病患者的死亡可归因于这一病原体。本实验室采用优化的基因定位方法，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、缺氧适应机制和吞噬抑制机制进行了系统筛选。然而，在这些筛选中发现的许多基因，虽然对致病性至关重要，但具有未知的分子功能。因此，获得对毒力的进一步了解需要能够导致新基因功能注释的方法。目的：利用化学遗传学分析这一强大的化学遗传学方法来解决功能注释问题。在这种方法中，化学扰动对大量已定义基因敲除的适应度的影响是量化的。由此产生的表型指纹被用来将基因聚类到功能组中，并确定它们彼此之间的作用。我们建议将这种方法应用于新形态C.对致病性所需的关键基因进行功能性注释。为了实现这一目标，我们将首先建立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未知功能基因产物在宿主病原体适应性中的作用的具体假设。影响：提出的工作将产生任何人类微生物病原体的大部分基因组的第一个详细的表型图。通过将具有相似特征的基因聚类在一起，预计这些研究将导致对先前在哺乳动物感染研究中确定的病原体因子的分子功能的迫切需要的深入了解。此外，这项工作有望确定基本毒力途径的化学调节剂，这有望成为病原体生物学研究和未来治疗发展的有力工具。

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. PUBLIC HEALTH RELEVANCE: The diagnosis and treatment of serious fungal infections of humans is a major unmet clinical challenge. Cryptococcus neoformans is among the most important fungal pathogens, accounting for approximately one-third of worldwide deaths from HIV/AIDS. By combining small molecules and genetics, this study will decipher the function of pathogen genes required for success in the host whose molecular functions are currently unknown. This knowledge is anticipated to produce a new foundation for the development of much-needed therapeutics.