Role of Coxiella burnetii group I introns in growth modulation

伯氏柯克斯体 I 组内含子在生长调节中的作用

基本信息

批准号：
7843521
负责人：
Michael F Minnick
金额：
$ 19.46万
依托单位：
UNIVERSITY OF MONTANA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-15 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7843521
关键词：
Acute Address Adenine Aerosols Antibodies Bacteria Biological Biological Assay Biology Buffers Catalytic RNA Cells Centers for Disease Control and Prevention (U.S.)Chronic Chronic Hepatitis Clinical Complex Coxiella Coxiella burnetii DNA Data Deoxyribonuclease I Dependency Development Disease Dose Electroporation Endocarditis Environment Enzymes Escherichia coli Event Exclusion Exons Figs - dietary Fostering Generations Genome Goals Growth Growth and Development function Guanosine Triphosphate Homing Human In Vitro Infection Interruption Introns Laboratories Luciferases Malaise Mediating Molecular Biology Mutate Nature Phase Play Pneumonia Production Proteins Q Fever RNA RNA Splicing Recombinant DNA Recombinants Relative (related person)Reporter Research Ribosomal RNA Ribosomes Role Site Testing Therapeutic Intervention Time Translations USSR Variant Virulence Western Blotting assay development cofactor design endonuclease expectation falls genetic element in vitro Assay in vivo mutant novel pathogen prevent public health relevance rRNA Genes transmission process two-dimensional weapons

项目摘要

DESCRIPTION (provided by applicant): Coxiella burnetii is an extremely infectious, obligate intracellular bacterium that causes Q fever, and is classified as a Select B Agent. Q fever is categorized as acute (pneumonitis, malaise) or chronic (hepatitis, endocarditis). Long-term goals are to identify bacterial factors that contribute to C. burnetii's developmental cycle, slow growth rate, and persistence in the environment. three interwoven virulence attributes. Preliminary data show that Coxiella's single 23S rDNA encodes two self-splicing, group I introns that inhibit ribosome function. We hypothesize that group I introns play central roles in C. burnetii's biology by: 1) helping maintain a slow growth rate, thereby fostering chronic infection, 2) promoting dormancy and environmental persistence by inhibiting translation as the bacterium transitions from a physiologically-active cell in the exponential phase (LCV) to a persistent, dormant-cell (SCV) during stationary phase, and 3) being involved in growth and development, since intron RNA (ribozyme) splicing is required to form mature 23S rRNA. Aim 1 will examine ribosome inhibition by ribozymes. Preliminary data show that both ribozymes associate with ribosomes of Coxiella or E. coli. Data also show that ribozymes retard growth of both bacteria. Aim 1 will examine ribozyme - ribosome interactions in detail to better understand the mechanisms involved. The first goal will build on preliminary data showing that electroporation of Cbu.L1917 RNA impairs C. burnetii growth. Dose-dependency and the possibility that Cbu.L1917 and Cbu.L1951 elicit differential levels of inhibition will be determined. A second goal will analyze the ribosome subunit targets of each ribozyme. Finally, the role of the internal guide sequence (IGS) in ribosome targeting will be analyzed with IGS mutants and wild-type introns in in vivo and in vitro assays. Aim 2 will elucidate the role of introns in development. Preliminary data suggest that ribozyme quantities are inversely proportional to Coxiella genomes during exponential phase. We will examine this relationship in long-term cultures to determine whether the opposite occurs as the cell approaches stationary phase. In addition to ribozyme-mediated inhibition of the ribosome, an alternative strategy is to produce fewer, functional 23S rRNAs by decreased splicing efficiency in stationary phase, and this possibility will be examined. Finally, we will determine whether exon skipping occurs; an event that would prevent mature 23S rRNA formation by exclusion of a 34-bp exon flanked by the two introns. Aim 3 will analyze unusual attributes of each intron. Cbu.L1917 (unique among group I introns in possessing a terminal &A instead of &G) will be assayed for self-splicing efficiency relative to a mutated intron with &G. Cofactors will be analyzed by varying the RNA splicing buffer. Two final goals will follow expression of a potential homing endonuclease (HE) encoded by Cbu.L1951, during development and assay HE for DNA endonuclease (homing) and maturase (splice facilitation) functions in vitro. This study will provide valuable information on the molecular biology of Coxiella and group I introns, and elucidate a possible target for therapeutic intervention of Q fever. PUBLIC HEALTH RELEVANCE: Coxiella burnetii is a zoonotic, bacterial pathogen that causes Q fever in humans and is classified as a select B agent by the CDC. The proposed research will investigate two genetic elements and associated events, hypothesized to contribute to C. burnetii's slow growth rate (fosters formation of chronic infection) and developmental cycle (provides for environmental persistence and enhanced transmission of the agent). In addition to helping address a dearth of information available on C. burnetii's molecular biology, interruption of these genetic elements and events will provide novel targets and strategies for therapeutic intervention of Q fever.

描述（由申请人提供）：伯内特柯克斯体是一种传染性极强的专性细胞内细菌，可引起 Q 热，并被归类为 Select B 病原体。 Q 热分为急性（肺炎、不适）或慢性（肝炎、心内膜炎）。长期目标是确定导致伯氏念珠菌发育周期、缓慢生长速率和在环境中持久存在的细菌因素。三种相互交织的毒力属性。初步数据显示，柯克斯体的单个 23S rDNA 编码两个自我剪接的 I 组内含子，可抑制核糖体功能。我们假设 I 组内含子在伯内特梭菌的生物学中发挥着核心作用：1）帮助维持缓慢的生长速度，从而促进慢性感染，2）当细菌从生理活性细胞转变时，通过抑制翻译来促进休眠和环境持久性3) 参与生长和发育，因为内含子 RNA（核酶）剪接是形成成熟 23S rRNA 所必需的。目标 1 将检查核酶对核糖体的抑制作用。初步数据表明，两种核酶均与柯克斯体或大肠杆菌的核糖体结合。数据还表明，核酶会阻碍这两种细菌的生长。目标 1 将详细检查核酶 - 核糖体相互作用，以更好地理解所涉及的机制。第一个目标将建立在初步数据的基础上，这些数据表明 Cbu.L1917 RNA 的电穿孔会损害伯内特氏菌的生长。将确定剂量依赖性以及 Cbu.L1917 和 Cbu.L1951 引起不同水平抑制的可能性。第二个目标将分析每个核酶的核糖体亚基靶标。最后，将在体内和体外测定中使用IGS突变体和野生型内含子来分析内部引导序列（IGS）在核糖体靶向中的作用。目标 2 将阐明内含子在发育中的作用。初步数据表明，核酶数量与指数期的柯克斯体基因组成反比。我们将在长期培养中检查这种关系，以确定当细胞接近稳定期时是否会发生相反的情况。除了核酶介导的核糖体抑制之外，另一种策略是通过降低稳定期的剪接效率来产生更少的功能性 23S rRNA，这种可能性将得到检验。最后判断是否发生外显子跳跃；通过排除两侧内含子的 34 bp 外显子来阻止成熟 23S rRNA 形成的事件。目标 3 将分析每个内含子的异常属性。将检测 Cbu.L1917（在 I 组内含子中唯一具有末端 &A 而不是 &G）相对于带有 &G 的突变内含子的自剪接效率。通过改变 RNA 剪接缓冲液来分析辅因子。在体外开发和检测 HE 的 DNA 核酸内切酶（归巢）和成熟酶（易剪接）功能期间，Cbu.L1951 编码的潜在归巢核酸内切酶 (HE) 的表达将实现两个最终目标。这项研究将为柯克斯体和 I 组内含子的分子生物学提供有价值的信息，并阐明 Q 热治疗干预的可能目标。公共卫生相关性：伯内特立克斯体是一种人畜共患的细菌病原体，可引起人类 Q 热，并被 CDC 列为精选 B 病原体。拟议的研究将调查两个遗传因素和相关事件，假设它们导致伯氏念珠菌缓慢的生长速度（促进慢性感染的形成）和发育周期（提供环境持久性和增强病原体的传播）。除了帮助解决伯氏念珠菌分子生物学信息的缺乏之外，中断这些遗传元件和事件还将为 Q 热的治疗干预提供新的靶点和策略。