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

脑膜炎病原体基因组的化学遗传学功能注释

• 批准号: 8448066
• 负责人: Hiten D Madhani
• 金额: $ 42.25万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8448066
• 关键词: AIDS/HIV problem; Accounting; Acquired Immunodeficiency Syndrome; Address; Animal Model; Animals; Biochemical; Biological; Biology; Cell physiology; Cells; Cessation of life; Chemicals; Clinical; Cluster Analysis; Collection; Communities; Cryptococcus neoformans; Data; Data Analyses; Databases; Development; Diagnosis; Disease; Dissection; Drug Formulations; Encapsulated; Equipment and supply inventories; FDA approved; Fingerprint; Foundations; Fungal Meningitis; Future; Gene Cluster; Gene Deletion; Gene Targeting; Generations; Genes; Genetic; Genome; Genomics; Goals; Growth; Human; Hypoxia; Individual; Industrial fungicide; Infection; Informatics; Knock-out; Knowledge; Laboratories; Lead; Libraries; Life; Maps; Measurement; Medical; Meningitis; Metabolism; Methods; Molecular; Mus; Mycoses; Nature; Pathogenicity; Pathway interactions; Patients; Phagocytosis Inhibition; Pharmaceutical Preparations; Process; Proteins; Reporting; Resolution; Resources; Robotics; Role; Saccharomyces cerevisiae; Saccharomycetales; Series; Signal Transduction; Testing; The science of Mycology; Therapeutic; Virulence; Virulence Factors; Work; Yeast Model System; Yeasts; chemical genetics; computerized data processing; density; design; effective therapy; fitness; follow-up; functional group; fungus; genome annotation; genome-wide; imaging modality; insight; knockout gene; microbial; novel; pathogen; relational database; response; small molecule; success; therapeutic development; tool

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.

描述（由申请人提供）： 理由：有效治疗人类危及生命的真菌感染是一个尚未解决的重大临床挑战。人类主要真菌病原体的注释基因组序列现已可用，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，由于模型酵母中的全基因组敲除集合有助于解剖基本的真核细胞过程，因此病原真菌中类似基因删除集合的生成和分析现在开始允许系统地阐明哺乳动物宿主毒力的分子决定因素。新型隐球菌是人类最重要的三种真菌病原体之一。它在实验上非常容易处理，具有适合遗传学的完整性周期和出色的感染动物模型。这种机会性封装芽殖酵母是真菌性脑膜炎的最常见原因。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种病原体。我们实验室采用优化的基因打靶方法，构建了1201个基因缺失菌株的文库。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系统筛选。然而，在这些筛选中鉴定出的许多基因虽然对致病性至关重要，但分子功能未知。因此，进一步了解毒力需要能够对新基因进行功能注释的方法。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）来解决功能注释问题。在这种方法中，化学扰动对大量确定的基因敲除的适应性的影响被量化。由此产生的表型指纹用于将基因聚类成功能组并定义它们彼此之间的作用。我们建议将此方法应用于新型隐球菌，以对致病性所需的关键基因进行功能注释。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，我们将获得并分析对一系列生物活性化学物质的完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关于未知功能的基因产物的作用的具体假设，我们之前已经将这些基因产物与宿主中的病原体适应性有关。影响：拟议的工作将产生任何人类微生物病原体大部分基因组的第一个详细表型图。通过将具有相似特征的基因聚集在一起，这些研究预计将有助于深入了解先前哺乳动物感染研究中确定的病原体因子的分子功能。此外，这项工作预计将定义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开发的强大工具。