Riboregulation in Pathogenic Rickettsiae

 描述(申请人提供)：康氏立克次体是地中海斑点热(MSF)的病原体，通过扁虱叮咬传播给人类，并优先感染血管系统内的微血管内皮细胞，导致“立克次体血管炎”。康氏立克次体与立克次体密切相关，立克次体引起落基山斑点热病流行。 美洲。致病性立克次体自然生命周期的一个耐人寻味但尚未被完全探索的特征是，它们能够成功地在变温硬蜱和 高度进化的哺乳动物，表明转录组在不同的宿主界面上的时空调节。尽管立克次体基因组很小，而且有还原进化的趋势，但立克次体适应不同寄主环境的机制(S)仍然几乎未知。最近，小调控RNA(SrRNAs)已成为细菌中最重要的转录后调控因子。其中，反式作用的srRNA通过RNA伴侣直接与靶基因(S)结合来发挥作用，而顺式作用的srRNA存在于开放阅读框架的反义链上。总之，这些srRNA通过转录的启动/终止、靶mRNAs的稳定/降解和翻译的调节来调节细菌的基因表达模式。我们利用生物信息学工具的组合，在圆顶红假单胞菌基因组中鉴定了33个srRNAs(包括6S RNA、β-tmRNA和RNase P)。我们进一步证明了至少4个新的候选基因，即srRNA1-srRNA4，在感染康氏杆菌的人微血管内皮细胞和壁虱载体细胞中都有表达，有趣的是，srRNA1和srRNA1(但不包括srRNA3和4)在人和壁虱细胞中的差异表达。这些发现提供了令人信服的基础数据，支持了我们的假设，即srRNA介导的转录组调控是立克次体应激反应、适应和毒力的重要决定因素，取决于它们的细胞内宿主生态位。因此，目标1建议使用差异RNA测序方法鉴定康氏立克次体中所有顺式和反式srRNAs，然后对新的立克次体srRNAs在哺乳动物宿主(HMECs)和壁虱载体(RSE8)细胞中的表达水平进行定量比较分析。值得注意的是，~50%的细菌中不存在已建立的RNA伴侣Hfq，但即使是那些缺乏Hfq的细菌也有功能的srRNA和新发现的RNA伴侣，如幽门螺杆菌中的HP1334。立克次体基因组中缺乏Hfq同源物，这意味着目前尚未确定的RNA伴侣(S)促进了srRNA-mR NA的相互作用。因此，目的2建议鉴定和鉴定与新的srRNA相互作用的辅助核糖核酸伴侣(S)，并确定其在罗氏酵母核转录调控中的作用。这项建议与美国国立卫生研究院的使命高度相关，因为它的目标是识别新的小调节RNA以及 通过应用最先进的基因组学和蛋白质组学方法，在NIAID C类优先病原体与宿主(人)和载体(TICK)细胞相互作用过程中的互补伴侣。

 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.