Uncovering small RNAs that contribute to Coxiella burnetii infection

发现导致伯内氏柯克斯体感染的小 RNA

• 批准号: 9529169
• 负责人: Rahul Raghavan
• 金额: $ 7.43万
• 依托单位: PORTLAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-19 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9529169
• 关键词: Acute; Aerosols; Affect; Anaplasma; Bacteria; Binding; Bioinformatics; Biological Assay; Biology; Cell physiology; Cells; Chlamydia; Chronic; Coxiella; Coxiella burnetii; Data; Development; Disease Outbreaks; Distant; Ehrlichia; Endocarditis; Enterobacteriaceae; Environment; Epidemic; Etiology; Gene Expression; Gene Expression Regulation; Genes; Genus staphylococcus; Goals; Goat; Growth; Human; Infection; Knowledge; Lead; Legionella; Livestock; Measures; Mediating; Membrane Proteins; Messenger RNA; Mitochondria; Molecular; Netherlands; Northern Blotting; Organism; Outcome; Pathogenesis; Pathogenicity; Process; Proteins; Public Health; Q Fever; Quantitative Reverse Transcriptase PCR; Regulation; Regulator Genes; Regulon; Research; Ribonucleases; Rickettsia; Role; Small RNA; Technology; Testing; Time; Translations; Treatment Efficacy; Untranslated RNA; Validation; Virulence; Work; Zoonoses; base; improved; innovation; insight; macrophage; monocyte; mutant; novel; novel therapeutic intervention; novel therapeutics; pathogen; prevent; targeted treatment; transcriptome; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT Non-coding small RNAs (sRNAs), which act swiftly and specifically, are ideal gene regulators in pathogens such as Coxiella burnetii that shift rapidly between disparate environments (e.g. aerosol vs. intracellular). However, very little is known about sRNAs that facilitate the pathogenicity of C. burnetii—a select agent that causes acute Q fever and chronic endocarditis. It is important that we gain better molecular insights into C. burnetii infection because it is found across the globe and has the potential to cause epidemics such as the recent outbreak in the Netherlands that involved more than 4,000 human cases and resulted in the culling of over 50,000 goats (a primary reservoir). Our long-term goal is to define the functions of non- coding RNAs during C. burnetii infection and to apply this knowledge to developing novel therapeutic ap- proaches. Towards attaining this goal, the objective of this application is to identify sRNAs that promote C. burnetii's growth within human cells. Based on preliminary data that identified several novel sRNAs in Cox- iella, our central hypothesis is that sRNAs promote intracellular growth of C. burnetii. The objectives of this project will be accomplished by two Specific Aims: (1) Identify sRNAs that are important to Coxiella's intra- cellular growth. Using RNA-seq we expect to identify sRNAs that are expressed during various stages of in- fection, and by examining transposon-insertion mutants we will assay their importance to intracellular growth. (2) Define the role of the sRNA CbsR14 in promoting Coxiella's intracellular growth. To begin to understand how sRNAs facilitate infection, we will interrogate the function of CbsR14, This sRNA was cho- sen because its expression was induced intracellularly, a transposon insertion in it significantly reduced Coxiella's intracellular growth, and preliminary analyses indicated that it targets the adjacent yidC gene. All studies to date on C. burnetii infection have only investigated proteins; hence, the research proposed here is innovative because it will depart from the status quo to investigate the role of non-coding RNAs in facilitat- ing Coxiella infection. This study is significant because it will, for the first time, identify sRNAs that pro- mote intracellular growth of C. burnetii. This new knowledge is expected to advance the field considerably by transforming our current understanding of Coxiella's pathogenicity. Additionally, uncovering the regula- tory circuit of YidC could be applicable to other organisms because this membrane protein is conserved across bacteria and mitochondria. Furthermore, the approaches we develop in this project will guide us (and others) to analyze other classes of non-coding RNAs (e.g. riboswitches) in Coxiella, and to examine sRNAs in other intractable intracellular pathogens such as Chlamydia, Rickettsia, Anaplasma and Ehrlichia. Moreover, a deeper understanding of sRNA-based gene regulation could contribute to the development of novel anti-Coxiella therapeutic agents that target critical components of the sRNA regulon.

项目总结/摘要 非编码小RNA（Non-coding small RNAs，sRNAs）是病原体中理想的基因调控因子，具有快速、特异的作用 例如贝氏柯克斯体（Coxiellaburnetii），其在完全不同的环境（例如气溶胶与细胞内）之间迅速转移。 然而，对促进C.选择性药剂 导致急性Q热和慢性心内膜炎重要的是我们能更好地了解分子 转化为C.贝氏感染，因为它是发现在地球仪，并有可能导致流行病， 最近在荷兰爆发的疫情涉及4,000多人病例， 扑杀50 000多只山羊（主要储存地）。我们的长期目标是确定非 在C.并将这些知识应用于开发新的治疗方法， 接近为了实现这一目标，本申请的目的是鉴定促进C. 伯内特氏菌在人体细胞中的生长基于在考克斯- iella，我们的中心假设是sRNAs促进了C.伯内特氏菌这一目标 本项目将通过两个具体目标来完成：（1）鉴定对柯克斯体内重要的sRNAs， 细胞生长使用RNA-seq，我们希望鉴定在不同阶段表达的sRNAs。 转染，并通过检查转座子插入突变体，我们将测定其重要性，细胞内 增长(2)定义sRNA CbsR 14在促进Coxiella细胞内生长中的作用。开始 为了了解sRNA是如何促进感染的，我们将探讨CbsR 14的功能，这种sRNA是选择性的。 因为它的表达是在细胞内诱导的，所以转座子插入它显著减少了 Coxiella的细胞内生长，初步分析表明，它的目标是相邻的yidC基因。所有 迄今为止，C. Burnetii感染只研究了蛋白质;因此，这里提出的研究是 创新，因为它将脱离现状，研究非编码RNA在促进中的作用， 柯克斯体感染。这项研究意义重大，因为它将首次鉴定出促生长的sRNAs。 C.伯内特氏菌这一新知识有望大大推进该领域的发展 通过改变我们目前对柯克斯体致病性的理解此外，发现规律- 由于YidC膜蛋白是保守的，因此YidC的保守回路也适用于其他生物 细菌和线粒体之间。此外，我们在这个项目中开发的方法将指导我们（和 其他）分析柯克斯体中其他类别的非编码RNA（例如核糖开关），并检查sRNA 在其他难治性细胞内病原体如衣原体、立克次体、无形体和埃里希体中。 此外，对基于sRNA的基因调控的更深入理解可能有助于 靶向sRNA调节子的关键组分的新型抗柯克斯体治疗剂。