Defining the functions of uncharacterized genes in priority pathogens

定义优先病原体中未表征基因的功能

• 批准号: 8719930
• 负责人: Sean Crosson
• 金额: $ 85.02万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-13 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8719930
• 关键词: Address; Affect; Animal Disease Models; Animal Model; Animals; Bacillus anthracis; Bacterial Proteins; Biochemical; Biochemistry; Biogenesis; Bioinformatics; Biological; Biological Assay; Biology; Biophysics; Brucella; Brucella abortus; Brucellosis; Categories; Cell physiology; Cells; Centers for Disease Control and Prevention (U.S.); Chicago; Chronic; Code; Collaborations; Communicable Diseases; Communities; Coxiella burnetii; DNA Sequence; Data; Databases; Disease; Fostering; Functional RNA; Genes; Genetic Transcription; Goals; Human; Immune; In Vitro; Inbred BALB C Mice; Individual; Infection; Instruction; Interdisciplinary Study; Investigation; Laboratories; Maintenance; Microbiology; Modeling; Molecular; Mus; National Institute of Allergy and Infectious Disease; Open Reading Frames; Operon; Oxidative Stress; PAWR protein; Pathogenesis; Physiological; Physiology; Principal Investigator; Process; Protein Family; Proteins; Publishing; Reagent; Regulation; Research; Research Infrastructure; Research Personnel; Research Project Grants; Resources; Role; Side; Sigma Factor; Signal Transduction; Stress; Structure; Technology; Testing; Transcriptional Regulation; Universities; Virulence; Yersinia pestis; acid stress; base; biological adaptation to stress; cell envelope; data management; gene function; genetic regulatory protein; genome sequencing; global health; in vivo; member; pathogen; programs; research study; success

DESCRIPTION (provided by applicant): Understanding the functions of the thousands of uncharacterized genes in sequence databases is among the most significant challenges in biology. The primary goal of this proposal is to define the biochemical and physiological functions of 235 uncharacterized genes in NIAID Category A (Bacillus anthracis and Yersinia pestis) and Category B (Brucella abortus and Coxiella burnetii) priority pathogens. These bacterial pathogens are highly infectious and have had a profound effect on global health both historically and currently. Included in this set are 185 predicted protein coding genes, 59 of which have no evidence of expression. The remaining 50 genes in our set are expressed, or are predicted to be expressed as small, non-coding RNAs (sRNAs). Our state-of-the-art research infrastructure at the Howard Taylor Ricketts Regional Biocontainment Laboratory (HTRL) and Argonne National Laboratory (ANL), and our integrated expertise in biochemistry and microbiology make us an ideal team for this interdisciplinary gene function assignment project. Our proposed research program will 1) yield important data on the function of uncharacterized genes in priority pathogens, 2) define the role of numerous uncharacterized genes in processes tied to bacterial pathogenesis, and 3) will produce useful biological and biochemical reagents for the pathogen research community. RELEVANCE: A full understanding of pathogen biology requires an understanding of how pathogen genes function at a biochemical level. Experiments proposed herein will define the biochemical function of uncharacterized genes implicated in disease processes in four NIAID priority pathogens. Project 1: Functional and Biochemical Characterization of B. abortus Stress Response Genes Project Leader: Sean Crosson DESCRIPTION: The immediate goal of this project is to define the cellular and biochemical functions of 57 uncharacterized open reading frames (ORFs), hypothetical proteins, and small non-coding RNAs in B. abortus. Our preliminary data provide evidence that these genes have a functional role in general stress adaptation and chronic mammalian infection. General stress response (GSR)-dependent transcription in B. abortus is controlled by the sigma factor SigE1. Our preliminary investigation of SigE1 and its upstream signaling partner PhyR have demonstrated that these regulatory proteins are required for adaptation to oxidative and acid stress in vitro and for maintenance of chronic infection in a BALB/c murine model. We have experimentally defined a set of 108 genes that are directly or indirectly regulated by SigE1. Within this regulated gene set are 32 uncharacterized open reading frames (ORFs) and nine small non-coding RNAs. Additionally, we have identified 16 hypothetical genes that are adjacent to or in apparent operons with SigE1-regulated ORFs but for which we have no evidence of expression. We propose to: 1) Test strains in which these genes have been deleted in oxidative and acid stress survival assays, and in cell-based and animal infection assays, 2) elucidate the biochemical functions of these hypothetical proteins and SigE1-regulated ORFs in collaboration with our Biochemical Function Technology Core, and 3) characterize the biochemical function of putative non-coding RNAs under transcriptional control of SigE1. These experiments will provide a comprehensive (in vivo and in vitro) functional understanding of conserved and non-conserved B. abortus genes that are currently uncharacterized. RELEVANCE: Brucella spp.are the causative agents of brucellosis, which is among the most common zooneses globally. Due to their high infectivity, easy aerosolization, and debilitating effects on infected individuals. Brucella spp have been classified as a category B bioterror threat by the U.S. Centers for Disease Control (CDC). Experiments proposed herein will define the biochemical function of uncharacterized genes implicated in regulation of B. abortus stress physiology and in the control of chronic brucellosis disease.

描述（由申请人提供）：了解序列数据库中数千个未表征基因的功能是生物学中最重大的挑战之一。该提案的主要目标是确定 NIAID A 类（炭疽杆菌和鼠疫耶尔森氏菌）和 B 类（流产布鲁氏菌和伯氏柯克斯体）优先病原体中 235 个未表征基因的生化和生理功能。这些细菌病原体具有高度传染性，在历史上和现在都对全球健康产生了深远的影响。该组中包括 185 个预测的蛋白质编码基因，其中 59 个没有表达证据。我们组中的其余 50 个基因已表达或预计将表达为小非编码 RNA (sRNA)。 我们在 Howard Taylor Ricketts 区域生物防护中心拥有最先进的研究基础设施 实验室 (HTRL) 和阿贡国家实验室 (ANL)，以及我们在生物化学和微生物学方面的综合专业知识，使我们成为这个跨学科基因功能分配项目的理想团队。我们提出的研究计划将1）产生关于优先病原体中未表征基因功能的重要数据，2）定义许多未表征基因在与细菌发病机制相关的过程中的作用，3）将为病原体研究产生有用的生物和生化试剂社区。 相关性：要充分了解病原体生物学，就需要了解病原体基因如何在生化水平上发挥作用。本文提出的实验将定义与四种 NIAID 优先病原体的疾病过程有关的未表征基因的生化功能。 项目1：流产布鲁氏菌应激反应基因的功能和生化特征 项目负责人：肖恩·克罗森 描述：该项目的直接目标是确定流产布鲁氏菌中 57 个未表征的开放阅读框 (ORF)、假设蛋白质和小非编码 RNA 的细胞和生化功能。我们的初步数据提供的证据表明这些基因在一般应激适应和慢性哺乳动物感染中具有功能性作用。流产布鲁氏菌中一般应激反应 (GSR) 依赖性转录受 sigma 因子 SigE1 控制。我们对 SigE1 及其上游信号传导伴侣 PhyR 的初步研究表明，这些调节蛋白是适应体外氧化和酸应激以及维持 BALB/c 小鼠模型中的慢性感染所必需的。我们通过实验定义了一组 108 个直接或间接受 SigE1 调控的基因。在这个受调控的基因集中有 32 个未表征的开放阅读框 (ORF) 和 9 个小非编码 RNA。此外，我们还鉴定了 16 个假设基因，这些基因与 SigE1 调节的 ORF 相邻或位于明显的操纵子中，但我们没有表达的证据。我们建议：1) 在氧化和酸应激生存测定以及基于细胞和动物感染测定中测试这些基因已被删除的菌株，2) 合作阐明这些假设蛋白和 SigE1 调节的 ORF 的生化功能使用我们的生化功能技术核心，3) 表征在 SigE1 转录控制下假定的非编码 RNA 的生化功能。这些实验将为目前尚未表征的保守和非保守流产布鲁氏菌基因提供全面的（体内和体外）功能理解。 相关性：布鲁氏菌属是布鲁氏菌病的病原体，布鲁氏菌病是全球最常见的人畜共患病之一。由于它们的传染性高，容易雾化，并对感染者产生衰弱作用。布鲁氏菌属已被列为 B 类生物恐怖威胁 由美国疾病控制中心 (CDC) 负责。本文提出的实验将确定涉及流产布鲁氏菌应激生理学调节和慢性布鲁氏菌病控制的未表征基因的生化功能。