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

• 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
• 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

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（由申请方提供）：贝氏柯克斯体是一种极具感染性的专性细胞内细菌，可引起Q热，被归类为选择B类病原体。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C. Burnetii的发育周期，缓慢的生长速度和在环境中的持久性。三个相互交织的毒力属性。初步数据显示，Coxiella的单一23 S rDNA编码两个自我剪接的I组内含子，抑制核糖体功能。我们推测I组内含子在C. Burnetii's biology by：1）有助于维持缓慢的生长速率，从而促进慢性感染，2）通过抑制细菌从指数期（LCV）的生理活性细胞转变为稳定期期间的持久休眠细胞（SCV）时的翻译来促进休眠和环境持久性，以及3）参与生长和发育，因为需要内含子RNA（核酶）剪接以形成成熟的23 S rRNA。目的1将研究核酶对核糖体的抑制作用。初步数据表明，这两种核酶都与Coxiella或E.杆菌数据还表明，核酶延缓两种细菌的生长。目的1将详细研究核酶-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C。贝氏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。第二个目标是分析每个核酶的核糖体亚基靶点。最后，内部指导序列（IGS）在核糖体靶向中的作用将与IGS突变体和野生型内含子在体内和体外测定中进行分析。目的2将阐明内含子在发育中的作用。初步数据表明，核酶的数量是成反比的柯克斯体基因组在指数期。我们将在长期培养中研究这种关系，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23 S rRNA，并且将检查这种可能性。最后，我们将确定是否发生外显子跳读;这一事件将通过排除两侧有两个内含子的34 bp外显子来阻止成熟23 S rRNA的形成。目标3将分析每个内含子的不寻常属性。将测定Cbu.L1917（在I组内含子中独特的是具有末端&A而不是&G）相对于具有&G的突变内含子的自剪接效率。将通过改变RNA剪接缓冲液来分析辅因子。两个最终目标将遵循由Cbu.L1951编码的潜在归巢核酸内切酶（HE）的表达，在开发和体外测定HE的DNA核酸内切酶（归巢）和成熟酶（剪接促进）功能期间。本研究将为柯克斯体和I组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：贝氏柯克斯体是一种引起人类Q热的人畜共患细菌病原体，被CDC归类为选择性B病原体。这项拟议中的研究将调查两个遗传因素和相关事件，假设有助于C。贝氏体的缓慢生长速度（促进慢性感染的形成）和发育周期（提供环境持久性和增强的病原体传播）。除了帮助解决缺乏关于C。Burnetii的分子生物学，中断这些遗传元件和事件将为Q热的治疗干预提供新的靶点和策略。

项目成果

Pentamidine inhibits Coxiella burnetii growth and 23S rRNA intron splicing in vitro.

Pentamidine 在体外抑制伯氏柯克斯体生长和 23S rRNA 内含子剪接。

• DOI: 10.1016/j.ijantimicag.2010.05.017
• 发表时间: 2010
• 期刊: International journal of antimicrobial agents
• 影响因子: 10.8
• 作者: Minnick,MichaelF;Hicks,LindaD;Battisti,JamesM;Raghavan,Rahul
• 通讯作者: Raghavan,Rahul

The Intervening Sequence of Coxiella burnetii: Characterization and Evolution.

伯内特立克次体的插入序列：特征和进化。

• DOI: 10.3389/fcimb.2016.00083
• 发表时间: 2016
• 期刊: Frontiers in cellular and infection microbiology
• 影响因子: 5.7
• 作者: Warrier,Indu;Walter,MathiasC;Frangoulidis,Dimitrios;Raghavan,Rahul;Hicks,LindaD;Minnick,MichaelF
• 通讯作者: Minnick,MichaelF