Dual RNA-seq of Orientia tsutsugamushi in HUVEC cells. Dual RNA-seq of Orientia tsutsugamushi in HUVEC cells

Emerging and neglected diseases pose challenges as their biology is frequently poorly understood, and genetic tools often do not exist to manipulate the responsible pathogen. Organism agnostic sequencing technologies offer a promising approach to understand the molecular processes underlying these diseases. Here we apply dual RNA-seq to Orientia tsutsugamushi (Ot), an obligate intracellular bacterium and the causative agent of the vector-borne human disease scrub typhus. Half the Ot genome is composed of repetitive DNA, and there is minimal collinearity in gene order between strains. Integrating RNA-seq, comparative genomics, proteomics, and machine learning, we investigated the transcriptional architecture of Ot, including operon structure and non-coding RNAs, and found evidence for wide-spread post-transcriptional antisense regulation. We compared the host response to two clinical isolates and identified distinct immune response networks that are up-regulated in response to each strain, leading to predictions of relative virulence which were confirmed in a mouse infection model. Thus, dual RNA-seq can provide insight into the biology and host-pathogen interactions of a poorly characterized and genetically intractable organism such as Ot. Overall design: RNA-seq of Orientia tsutsugamushi strains UT176 and Karp in HUVEC cells with uninfected controls

新发与被忽视的传染病常面临严峻挑战：其致病生物学机制往往尚未明确，且缺乏可操控其致病病原体的遗传工具。不依赖物种的测序技术为解析此类疾病的分子致病过程提供了极具前景的研究路径。本研究针对恙虫病东方体（Orientia tsutsugamushi, Ot）开展双转录组测序（dual RNA-seq）：该菌为专性胞内寄生菌，是虫媒传播人类疾病——恙虫病（scrub typhus）的病原体。恙虫病东方体的基因组近半数由重复DNA序列组成，且不同菌株间的基因共线性极低。本研究整合转录组测序、比较基因组学、蛋白质组学与机器学习技术，解析了恙虫病东方体的转录组架构，包括操纵子结构与非编码RNA，并发现了广泛存在的转录后反义调控证据。本研究对比了宿主对两株临床分离株的应答反应，鉴定出两菌株分别诱导上调的独特免疫应答网络，据此预测了其相对毒力，并通过小鼠感染模型验证了该预测结果。由此可见，双转录组测序可帮助解析如恙虫病东方体这类特征匮乏且遗传操作困难的病原体的生物学特性及其与宿主的互作机制。
实验设计：对人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVEC）中感染恙虫病东方体UT176与Karp菌株的样本及未感染对照样本开展转录组测序。