Riboregulation in Pathogenic Rickettsiae

 描述（由申请方提供）：康氏立克次体是地中海斑点热（MSF）的病原体，通过蜱叮咬传播给人类，并优先感染血管系统内衬的微血管内皮，导致“立克次体血管炎”。R. conorii与R.立克次氏体，导致落基山斑疹热流行， 美洲。致病性立克次体自然生命周期的一个有趣但尚未完全探索的特征是它们能够成功地在变温蜱和 高度进化的哺乳动物，这表明时空调控的转录组在不同的主机接口。尽管立克次体的基因组很小，而且有还原进化的趋势，但立克次体适应不同宿主环境的机制实际上仍不清楚。最近，小调控RNA（srRNA）已成为细菌中最重要的转录后调节因子。其中，反式作用srRNA通过RNA伴侣直接结合靶mRNA来实现其作用，而顺式作用srRNA存在于开放阅读框的反义链上。这些srRNA共同通过转录的起始/终止、靶mRNA的稳定/降解以及翻译的调节来调节细菌基因表达模式。我们利用生物信息学的方法，在红毛菊中鉴定了33种srRNA（包括6S RNA、<$-tmRNA和RNase P）。conorii基因组。我们进一步证明了至少4种新的候选物，即srRNA 1-srRNA 4，在感染了R. conorii和有趣的是，srRNA 1和（但不是srRNA 3和4）在人类与蜱细胞中的差异表达。这些发现产生令人信服的基础数据支持我们的假设，即srRNA介导的转录组调控是立克次体应激反应，适应和毒力取决于其细胞内宿主生态位的重要决定因素。因此，目标1提出鉴定R中所有的顺式和反式作用srRNA。使用差异RNA测序，随后定量比较分析哺乳动物宿主（HMEC）和蜱载体（RSE 8）细胞中新型立克次体srRNA的表达水平。值得注意的是，在约50%的细菌中不存在已建立的RNA分子伴侣Hfq，但即使是那些缺乏Hfq的细菌也具有功能性srRNA和新发现的RNA分子伴侣，如H.幽门。立克次体基因组中缺乏hfq同源物，这表明尚未鉴定的RNA伴侣促进srRNA-mRNA相互作用。因此，目的2提出鉴定和表征与新srRNA相互作用的辅助RNA伴侣，并确定其对R.科诺里。这项提议与NIH的使命高度相关，因为它旨在鉴定新的小调控RNA以及一种新的调控RNA。 通过应用基因组学和蛋白质组学的最新方法，在NIAID C类优先病原体与宿主（人）和载体（蜱）细胞相互作用期间，在NIAID C类优先病原体中的互补分子伴侣。

 DESCRIPTION (provided by applicant): Rickettsia conorii, the etiological agent of Mediterranean spotted fever (MSF), is transmitted to humans by tick bite and preferentially infects microvascular endothelium lining the vasculature leading to `rickettsial vasculitis'. R. conorii is closely related to R. rickettsii, which causes Rocky Mountain spotted fever prevalent in the Americas. An intriguing, but as yet completely unexplored, feature of the natural lifecycle of pathogenic rickettsiae is their ability to successfully transition between poikilothermic ticks and highly evolved mammals, suggesting spatiotemporal regulation of the transcriptome at various host interfaces. In spite of small genomes and the tendency for reductive evolution, the mechanism(s) through which rickettsiae adapt to varying host environments remain virtually unknown. Recently, small regulatory RNAs (srRNAs) have emerged as the most important post-transcriptional regulators in bacteria. Among these, trans-acting srRNAs implement their effects by directly binding to target mRNA(s) through an RNA chaperone, whereas cis-acting srRNAs are present on the anti-sense strand of an open reading frame. Together, these srRNAs regulate bacterial gene expression patterns via initiation/termination of transcription, stabilization/degradation of target mRNAs, and regulation of of translation. We have utilized a combination of bioinformatics tools to identify a total of 33 srRNAs (including 6S RNA, ¿-tmRNA, and RNase P) in the R. conorii genome. We further demonstrate expression of at least 4 novel candidates, namely srRNA1-srRNA4, in both human microvascular endothelial cells and tick vector cells infected with R. conorii and intriguingly, differential expression of srRNA1 and (but not srRNA3 and 4) in human versus tick cells. These findings yield convincing foundation data in support of our hypothesis that srRNA-mediated regulation of transcriptome is an important determinant of rickettsial stress response, adaptation, and virulence depending on their intracellular host niches. Accordingly, Aim 1 proposes to identify all cis- and trans-acting srRNAs in R. conorii using differential RNA sequencing, followed by quantitative comparative analysis of the expression levels of novel rickettsial srRNAs in mammalian host (HMECs) and tick vector (RSE8) cells. Notably, an established RNA chaperone Hfq is absent in ~50% of bacteria, yet even those lacking Hfq have functional srRNAs and newly discovered RNA chaperones such as HP1334 in H. pylori. Rickettsiae lack hfq homologs in their genomes, implicating that as yet unidentified RNA chaperone(s) facilitate srRNA-mRNA interactions. Aim 2, therefore, proposes to identify and characterize an auxiliary RNA chaperone interacting with novel srRNA(s) and define its contributions to riboregulation in R. conorii. This proposal is highl relevant to the mission of the NIH, as it aims to identify novel small regulatory RNAs as well as a complementary chaperone in an NIAID category C priority pathogen during its interactions with the host (human) and vector (tick) cells by application of state-of-the-art approaches of genomics and proteomics.