Small Regulatory RNAs of Coxiella burnetii - The Agent 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细胞内细菌，可引起人体Q热，被归类为选择性病原体。Q热通常表现为一种使人衰弱的流感样疾病，伴有肺炎或肝炎， 但在少数情况下，严重的慢性感染会发生危及生命的心内膜炎， 主要表现。关于Coxiella的毒力决定因素或细菌如何调节与其双相生命周期和宿主细胞感染相关的事件， 尽管这些活动在生存中发挥着核心作用。我们的长期目标是阐明 RNA介导的这些过程的调节。我们小组的初步研究已经开始分析病原体的大细胞变体（LCV;稳定期）和小细胞变体（SCV;指数期）的小RNA（sRNA），我们已经确定6S RNA是一种突出的sRNA，其在SCV中的表达水平比LCV高约7倍。我们推测sRNAs在调节柯克斯体的生长、发育和感染宿主细胞中起关键作用。这些假设将在两个具体目标中得到解决。在目标1中， 通过使用高通量（深度）测序技术表征SCV和LCV的sRNA谱，鉴定参与调节Coxiella生长和发育的sRNA。其次，我们将通过鉴定和比较在宿主细胞中生长的细菌的sRNA与从纯性（无宿主细胞）培养物中获得的sRNA来分析柯克斯体的“感染特异性”sRNA。目的1的结果将为构建柯克斯体sRNA网络和阐明它们如何调控发育和胞内寄生提供基础。目的二是研究6S sRNA对柯克斯体生长发育的调控作用。6S RNA不是密切相关的嗜肺军团菌中稳定期基因的主要阻遏物，相反，6S RNA正调控几个增强细胞内生长和毒力的基因。这一观察结果，连同一份报告显示，SS是一个占主导地位的调节指数增长的柯克斯体，导致我们假设，6S sRNA调节DNA依赖的RNA聚合酶与西格玛70（RNAP-s70）和RNAP-SS，后者的复杂性的偏好。为了解决这一假设，我们将阐明柯克斯体的6S RNA与RNAP的相互作用，在分子水平上的个人S因子，RNAP-s70和RNAP-ss，通过生物化学和凝胶阻滞研究。其次，我们将进行RNA-seq转录组分析，以确定在6S RNA水平过量或降低时差异表达的柯克斯体基因。6S RNA水平将通过转录6S有义或反义RNA的稳定穿梭载体来操纵。6S（反义）正义RNA的表达将被载体上的顺式lacIQ基因抑制，并用IPTG诱导。RNA-seq结果将使我们能够制定一个6S RNA调控网络，并改善目前关于柯克斯体全球转录调控的知识水平。此外，这些结果将阐明6S sRNA在g-变形菌中的作用，其中直链因子在不同的细菌物种中发挥独特的作用。

DESCRIPTION (provided by applicant): Coxiella burnetii is an extremely infectious, intracellular bacterium that causes Q fever in humans and is classified as a select agent. Q fever typically presents as a debilitating, flu-like illness accompanied by pneumonia or hepatitis, but in a minority of cases a severe, chronic infection occurs with life-threatening endocarditis as the predominant manifestation. Little is known about Coxiella's virulence determinants or how the bacterium regulates events associated with its biphasic life cycle and infection of host cells, despite the central role that these activities play in survival. Our long-term goal is to elucidate the RNA-mediated regulation of these processes. Preliminary studies by our group have begun to analyze small RNAs (sRNAs) of large-cell variants (LCVs; stationary phase) and small-cell variants (SCVs; exponential phase) of the pathogen, and we have identified 6S RNA as a prominent sRNA that it is expressed at levels that are ~7-fold higher in SCVs relative to LCVs. We hypothesize that sRNAs play key roles in regulating Coxiella's growth, development and infection of host cells. These hypotheses will be addressed in two specific aims. In aim 1, we will identify sRNAs involved in regulating Coxiella's growth and development by characterizing sRNA profiles of SCVs and LCVs using high-throughput (deep) sequencing technology. Second, we will analyze "infection-specific" sRNAs of Coxiella by identifying and comparing sRNAs of bacteria grown in the context of host cells to those obtained from axenic (host cell-free) cultures. Results of aim 1 will provide a foundation to formulate Coxiella's sRNA networks and elucidate how they regulate development and intracellular parasitism. In aim 2, we will investigate the regulatory role of 6S sRNA in Coxiella's growth and development. 6S RNA is not a major repressor of stationary-phase genes in closely-related Legionella pneumophila, rather, the 6S RNA positively regulates several genes that enhance intracellular growth and virulence. This observation, together with a report showing that ss is a dominant regulator during exponential growth of Coxiella, leads us to hypothesize that 6S sRNA regulates both DNA-dependent RNA polymerase associated with sigma-70 (RNAP-s70) and RNAP-ss, with a preference for the latter complex. To address this hypothesis, we will elucidate Coxiella's 6S RNA interactions with RNAP, the individual s factors, RNAP-s70 and RNAP-ss at the molecular level, by biochemical and gel retardation studies. Second, we will conduct RNA-seq transcriptome analyses to identify Coxiella genes that are differentially expressed in response to excess or reduced levels of 6S RNA. The 6S RNA levels will be manipulated by stable shuttle vectors that transcribe 6S sense or antisense RNAs. Expression of 6S (anti)sense RNA will be repressed by a cis-lacIQ gene on the vector and induced with IPTG. RNA-seq results will allow us to formulate a 6S RNA regulatory network and improve the current state of knowledge regarding global transcriptional regulation in Coxiella. Moreover, the results will clarify the rol of 6S sRNA in g-proteobacteria, where orthologous s factors play distinctive roles in different bacterial species.