Novel Bacterial Small RNAs as Determinants of Rickettsial Virulence and Transmission

项目总结/摘要 致病性立克次体物种包括已知引起人类感染的革兰氏阴性细菌，即 斑点热（R. rickettsii和R. conorii）和斑疹伤寒（R. prowazekii和R.伤寒），发病率高， mortality.嗜微血管内皮细胞，快速逃离吞噬体，和生长/复制 营养丰富的胞质溶胶作为专性细胞内寄生虫导致血管炎症， 功能障碍/损伤构成立克次氏体发病机制的显著特征。立克次体病是媒介传播的 由受感染的节肢动物（包括蜱虫和跳蚤）在自然界传播的感染。立克次体能够 在自然媒介中通过经卵巢（水平）和经卵巢（垂直）传播传播， 在其生命周期中经历戏剧性的小生境特异性转录组适应，以适应不同的 环境中的节肢动物载体和哺乳动物宿主。在这种情况下，我们现有的一个主要真空 知识是缺乏对支持其适应性和/或致病性的调节机制的理解 在不同宿主生态位中的能力。虽然小的非编码RNA（sRNA）现在已经被牢固地确立为 病原菌的普遍特征及其转录组的主要调节因子， 立克次体sRNA的功能特征直到最近才被忽视。我们现在已经建立了， 发表了立克次体物种中的sRNA不仅丰富和功能（基于对 RC 0877和cydA分别作为新R. Conorii sRNA Rc_sR35和Rc_sR42，但 在人与蜱宿主细胞中以及在小鼠模型中的靶器官中也有差异表达， 内皮靶立克次体病类似于人类疾病。这些发现是一部小说的基础 我们假设致病性立克次体利用sRNAs网络适应 宿主特异性环境（载体维斯哺乳动物）通过基因表达的调节。建立了 sRNAs在两大致病性立克次体中的存在，我们现在建议进行一项 全面的“比较和对比”分析R. conorii和R.伤寒及其靶点 基因感染人类和小鼠微血管内皮细胞，并执行机制和 非常重要的立克次体sRNA的功能表征[目的1]。接下来，我们将研究它们在以下方面的作用： 立克次体在自然媒介中的维持和媒介-宿主传播 [Aim二、 并作为潜在的决定因素 在已建立的鼠R. conorii和R.伤寒感染密切相关 人类立克次体病的病理生理学概述 [Aim三、 .最后，建议的科学 通过对新立克次体sRNAs的编目和测定， 在不同的生命周期阶段， 维持、发病机制和传播。由此获得的知识将为以下方面提供一个平台： 战略性开发新的和改进的人类立克次体病治疗方法。

Project Summary/Abstract Pathogenic Rickettsia species include Gram-negative bacteria known to cause human infections, namely spotted fever (R. rickettsii and R. conorii) and typhus (R. prowazekii and R. typhi), with serious morbidity and mortality. Tropism for microvascular endothelium, rapid escape from the phagosome, and growth/replication in the nutrient-rich cytosol as obligate intracellular parasites leading to vascular inflammation and dysfunction/damage constitute the salient features of rickettsial pathogenesis. Rickettsioses are vector-borne infections transmitted in nature by infected arthropods, including ticks and fleas. Rickettsiae are capable of circulating in their natural vectors through transstadial (horizontal) and transovarial (vertical) transmission and undergo dramatic niche-specific transcriptomic adaptations during their life-cycle to adjust to diverse environments in arthropod vectors and mammalian hosts. In this context, a major vacuity in our existing knowledge is the lack of understanding of regulatory mechanisms supporting their adaptive and/or pathogenic abilities in different host niches. Although small non-coding RNAs (sRNAs) are now firmly established as universal features of pathogenic bacteria and major regulators of their transcriptomes, the discovery and functional characterization of rickettsial sRNAs remained ignored until recently. We have now established and published that sRNAs in Rickettsia species are not only plentiful and functional (based on the identification of RC0877 and cydA as the respective target genes for novel R. conorii sRNAs Rc_sR35 and Rc_sR42, but are also differentially expressed in human versus tick host cells and in target organs in a mouse model of endothelial-target rickettsiosis akin to human disease. These findings serve as the foundation for a novel paradigm and genesis of our hypothesis that pathogenic rickettsiae exploit a network of sRNAs to adapt to host-specific environments (vectors vis-á-vis mammals) via modulation of gene expression. Having established the presence of sRNAs in both major groups of pathogenic rickettsiae, we now propose to conduct a comprehensive `compare and contrast' analysis of sRNA repertoire of R. conorii and R. typhi and their target genes during infection of human and mouse microvascular endothelium and to perform mechanistic and functional characterization of critically important rickettsial sRNAs [Aim 1]. We will next investigate their roles in rickettsial maintenance in natural vectors and vector-to-host transmission [Aim 2] and as potential determinants of virulence in the target organs of established murine models of R. conorii and R. typhi infection closely recapitulating the pathophysiology of human rickettsioses [Aim 3] . At conclusion, the proposed scientific enquiry will have a sustained positive impact through cataloging of novel rickettsial sRNAs and determination of mechanistic and functional insights into transcriptome regulation during distinct life-cycle phases of maintenance, pathogenesis, and transmission. The knowledge thus acquired will provide a platform for strategic development of new and improved treatment modalities against human rickettsial diseases.