Uncovering small RNAs that contribute to Coxiella burnetii infection

项目摘要/摘要 非编码小RNA(SRNAs)作用迅速、特异性强，是病原体理想的基因调控因子 例如，伯氏柯克斯体在不同的环境之间迅速变化(例如，气溶胶与细胞内)。 然而，人们对促进伯氏梭菌致病的srna知之甚少。 会引起急性Q热和慢性心内膜炎。重要的是我们获得了更好的分子洞察力 导致伯氏梭菌感染，因为它在全球各地都有发现，并有可能导致诸如 由于最近在荷兰爆发的疫情涉及4000多例人感染病例，并导致 扑杀50,000多只山羊(主要蓄水池)。我们的长期目标是界定非政府组织的功能 编码伯氏梭菌感染过程中的RNA，并将这一知识应用于开发新的治疗AP。 行尸走肉。为了实现这一目标，本应用程序的目标是识别促进C。 伯氏杆菌在人体细胞内的生长。根据初步数据，在COX-1中发现了几个新的sRNA- Iella，我们的中心假设是sRNA促进了伯氏梭菌的细胞内生长。这样做的目的是 该项目将通过两个具体目标来完成：(1)确定对Coxiella内部的重要sRNA。 细胞生长。利用rna-seq技术，我们有望鉴定在不同发育阶段表达的sRNA。 通过检测转座子插入突变体，我们将分析它们对细胞内的重要性 成长。(2)明确SRNA CbsR14在促进柯克斯体细胞内生长中的作用。开始，开始 了解sRNA是如何促进感染的，我们将询问CbsR14的功能，这个sRNA是 因为它的表达是在细胞内诱导的，所以转座子的插入显著减少了 Coxiella的细胞内生长，初步分析表明它针对邻近的yidC基因。全 到目前为止，关于伯氏梭菌感染的研究只研究了蛋白质；因此，这里提出的研究是 创新是因为它将脱离现状，研究非编码RNA在促进- 柯克斯体感染。这项研究具有重要意义，因为它将首次识别促进... 伯氏隐翅虫的胞内微尘生长。这一新知识有望极大地推动该领域的发展。 通过改变我们目前对柯克斯体致病性的理解。此外，揭开规则- YidC的Tory环路可能适用于其他生物，因为该膜蛋白是保守的 跨越细菌和线粒体。此外，我们在此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 在其他难治的细胞内病原体中，如衣原体、立克次体、无浆体和埃立克体病。 此外，对基于sRNA的基因调控的更深入的理解可能有助于发展 针对sRNA调节子关键成分的新型抗柯克斯体治疗剂。

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.