Role of Coxiella burnetii group I introns in growth modulation

描述（由申请人提供）：伯纳氏杆菌是一种极具传染性的专性细胞内细菌，可引起Q热，被归类为精选B剂。Q热分为急性（肺炎、不适）和慢性（肝炎、心内膜炎）。长期目标是确定细菌因素，有助于伯纳蒂梭菌的发育周期，缓慢的生长速度，并在环境中持久性。三个相互交织的毒力属性。初步数据显示，Coxiella的单个23S rDNA编码两个自剪接的I族内含子，抑制核糖体功能。我们假设I组内含子在伯氏梭菌的生物学中起着核心作用：1)帮助维持缓慢的生长速度，从而促进慢性感染；2)当细菌从指数期（LCV）的生理活跃细胞转变为静止期的持续休眠细胞（SCV）时，通过抑制翻译促进休眠和环境持久性；3)参与生长和发育，因为需要内含子RNA（核酶）剪接才能形成成熟的23S rRNA。目的1将检验核糖体对核酶的抑制作用。初步数据表明，这两种核酶都与科希氏菌或大肠杆菌的核糖体结合。数据还显示，核酶延缓了这两种细菌的生长。目的1将详细检查核酶-核糖体相互作用，以更好地了解所涉及的机制。第一个目标将建立在显示Cbu电穿孔的初步数据上。L1917 RNA损害burnetii生长。剂量依赖性和Cbu。L1917和Cbu。L1951引起的不同程度的抑制将被确定。第二个目标是分析每个核酶的核糖体亚基靶标。最后，利用IGS突变体和野生型内含子进行体内和体外实验，分析内部引导序列（IGS）在核糖体靶向中的作用。目的2将阐明内含子在发育中的作用。初步数据表明，在指数阶段，核酶的数量与科希氏菌基因组成反比。我们将在长期培养中检查这种关系，以确定当细胞接近固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外，另一种策略是通过降低固定期剪接效率来产生更少的功能性23S rrna，这种可能性将被研究。最后，我们将确定是否发生外显子跳变；通过排除两个内含子两侧的34 bp外显子来阻止成熟的23S rRNA形成。目的3将分析每个内含子的不寻常属性。Cbu。L1917（在I组内含子中是独一无二的，它拥有末端&A而不是&G）将被用于检测相对于带有&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。两个最终目标将遵循Cbu编码的潜在归巢内切酶（HE）表达。1951年，在开发和测定DNA内切酶（归巢）和成熟酶（剪接促进）功能的体外HE。本研究将为科希氏菌和I族内含子的分子生物学研究提供有价值的信息，并阐明Q热治疗干预的可能靶点。公共卫生相关性：伯纳蒂克希菌是一种人畜共患的细菌性病原体，可引起人类Q热，被美国疾病控制与预防中心列为B型病原体。拟议的研究将调查两个遗传因素和相关事件，假设它们有助于伯纳蒂胞杆菌的缓慢生长速度（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）。除了有助于解决伯纳蒂胞杆菌分子生物学信息匮乏的问题外，这些遗传因素和事件的中断将为Q热的治疗干预提供新的靶点和策略。

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.