Defining the functions of uncharacterized genes in priority pathogens

描述(由申请人提供)：理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。这项建议的主要目标是确定NIAID A类(炭疽芽孢杆菌和鼠疫杆菌)和B类(流产布鲁氏菌和伯氏柯克斯体)优先病原体中235个未鉴定基因的生化和生理功能。这些细菌病原体具有很高的传染性，从历史上和现在都对全球健康产生了深远的影响。这一组包括185个预测的蛋白质编码基因，其中59个没有表达的证据。我们组中的其余50个基因表达或预测表达为小的非编码RNA(SRNAs)。 我们在霍华德·泰勒·里基茨区域生物遏制中心最先进的研究基础设施 我们的实验室(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，使我们成为这一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)确定大量未表征基因在与细菌发病相关的过程中的作用，以及3)将为病原体研究社区生产有用的生物和生化试剂。 相关性：全面了解病原体生物学需要了解病原体基因如何在生化水平上发挥作用。本文提出的实验将确定四种NIAID优先病原体中涉及疾病过程的未表征基因的生化功能。 项目1：流产杆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：该项目的近期目标是确定流产杆菌中57个未鉴定的开放阅读框架(ORF)、假设的蛋白质和小的非编码RNA的细胞和生化功能。我们的初步数据提供了证据，证明这些基因在一般压力适应和慢性哺乳动物感染中具有功能作用。一般应激反应(GSR)依赖的转录在流产杆菌中由Sigma因子SigE1控制。我们对SigE1及其上游信号伙伴PHYR的初步研究表明，这些调节蛋白在体外对氧化和酸应激的适应以及在BALB/c小鼠模型中维持慢性感染是必需的。我们已经通过实验定义了一组由SigE1直接或间接调控的108个基因。在这个受调控的基因集中，有32个未鉴定的开放阅读框架(ORF)和9个小的非编码RNA。此外，我们已经确定了16个假设的基因，这些基因与SigE1调节的ORF相邻或位于明显的操纵子中，但我们没有证据表明它们的表达。我们建议：1)在氧化和酸应激生存分析中，以及在基于细胞和动物感染的分析中，对这些基因缺失的菌株进行测试；2)与我们的生化功能技术核心合作，阐明这些假想的蛋白质和SigE1调节的ORF的生化功能；以及3)表征SigE1转录控制下假定的非编码RNA的生化功能。这些实验将提供一个全面的(在体内和体外)对保守和非保守的流产杆菌基因的功能理解，这些基因目前还没有特征。 相关性：布鲁氏菌是布鲁氏菌病的病原体，布鲁氏菌病是全球最常见的动物之一。由于它们的高传染性，容易气雾化，并对受感染的人产生衰弱作用。布鲁氏菌被列为B类生物恐怖威胁 由美国疾病控制中心(CDC)提供。本文提出的实验将确定未知基因的生化功能，这些基因与调节流产杆菌应激生理和控制慢性布鲁氏菌病有关。

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.