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

基本信息

批准号：
8823727
负责人：
Hiten D Madhani
金额：
$ 44.95万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-01 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8823727
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 Genetic study 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

项目摘要

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：有效治疗人类威胁生命的真菌感染是一项重大的未经临床挑战。现在可以使用主要人类真菌病原体的注释基因组序列，从而通过应用系统的方法创造了彻底改变医学真菌学的机会。特别是，由于模型酵母中全基因组的敲除收集对基本真核生物细胞过程的解剖至关重要，因此，致病真菌中类似基因缺失的产生和分析现在开始允许对哺乳动物宿主的病毒性分子确定性的系统阐明。隐孢子虫是人类中三种最重要的人类真菌病原体之一。它在实验上是高度可观的，具有完整的性周期，适合于遗传学和杰出的感染动物模型。这种机会性封装的萌芽酵母是真菌脑膜炎的最常见原因。每年，估计有1,000,000例导致约60万例死亡，艾滋病患者死亡中有三分之一归因于这种病原体。使用优化的基因靶向方法，我们的实验室构建了库1201基因缺失菌株。我们利用了该资源来用于实验小鼠中病原体适应性的系统筛查，已知毒力因子的表达，低氧适应机制以及抑制吞噬作用的机制。但是，在这些筛选中鉴定出的许多基因虽然对致病性至关重要，但具有未知的分子功能。因此，获得进一步的毒力，需要可以导致新基因功能注释的方法。目的：我们建议通过应用化学遗传分析（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基因敲除的适应性的影响进行了量化。所得的表型指纹用于将基因聚集到官能团中，并彼此定义它们的作用。我们建议将此方法应用于新生梭菌，以在功能上注释致病性所需的关键基因。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特征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品阵列的全稀释定量适应性响应。最后，我们将利用结果来开发和检验混凝土假设，以实现未知功能的基因产物的作用，我们以前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的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，预计这些研究将导致对OU先前对哺乳动物感染研究中鉴定的病原体因子的分子功能的急需见解。此外，预计这项工作将定义基本毒力途径的化学调节剂，预计这将是研究病原体生物学和未来治疗性发育的强大工具。