Elucidating the Rickettsia prowazekii EnvZ/OmpR Regulon

描述(申请人提供)：立克次体属成员是专性胞质内病原体，可导致人类流行和地方性斑疹伤寒等疾病。据估计，流行性斑疹伤寒的虱媒病媒普罗瓦泽克杆菌在上个世纪造成了300多万人死亡！Prowazekii是一种选择剂，已被NIAID指定为B类优先病原体。在体内，立克次体局限于有限数量的环境中，包括从虱子载体的肠道上皮细胞生长到人类宿主的血液和人类内皮细胞之间的过渡。考虑到已经成功适应在真核细胞胞质中生长的生物的稀缺性，胞质可以被认为是一个不利的环境。此外，由于这种固有的细胞内生活方式的性质，在非生长条件下在宿主细胞外度过的任何时间都是一种潜在的应激条件，表明与环境沟通的能力可能是普罗瓦泽克杆菌致病的关键。这项R21提案将为进一步探讨这些极其有趣的问题奠定基础。基于双组分反应调节因子在感知环境变化中的重要作用，我选择了普氏杆菌RP426/RP427(注释为EnvZ/OmpR)作为潜在的传感蛋白/反应调节因子对，并将阐明它们控制下的基因的调控。R21提案的目标是将这一双组分网络作为相关的生物学模型，使染色质免疫沉淀(CHIP)技术适用于专性细胞内、胞浆受限的病原体。具体目标描述了两种方法。我将使用纯化的、重组的OmpR、磷酸化的OmpR(OmpR约为P)和普罗瓦泽克杆菌的染色体DNA片段进行体外芯片检测。OmpR/OmpR大约P结合的调控序列将被分离、克隆，并通过DNA测序进行鉴定。这种体外方法将识别不受环境条件影响的调控序列。第二种方法是体内芯片分析，它将从受控环境条件下生长的普罗瓦泽克酵母中分离出调控DNA序列。这些条件将根据体外芯片产生的数据以及立克次体暴露的有限环境生态位来选择。这两种技术的结合将阐明由OmpR控制的普罗瓦泽克乳杆菌基因，并可能为该系统对环境刺激做出反应提供洞察力。与R21应用的探索性/发展性相一致，本研究是首次以普罗瓦泽克杆菌为模型系统，发展染色质免疫沉淀，以阐明专性细胞内病原体中的基因调节子。了解基因调控在发病机制中的作用可能是开发针对专性细胞内选择药物的对策和新疗法的关键。

DESCRIPTION (provided by applicant): Members of the genus Rickettsia are obligate intracytoplasmic pathogens that cause diseases such as epidemic and endemic typhus in humans. It is estimated that R. prowazekii, the louse-vectored agent of epidemic typhus, has caused more than 3 million deaths in the last century! R. prowazekii is a Select Agent and has been designated by the NIAID as a Category B Priority Pathogen. In vivo, rickettsiae are confined to a limited number of environmental niches that include transitioning between growth in gut epithelial cell of the louse vector to the blood stream of the human host and to the human endothelial cell. Considering the paucity of organisms that have successfully adapted to growth in eukaryotic cytosol as a growth niche, cytosol could be thought of as a hostile environment. In addition, due to the nature of this obligate intracellular life style, any time spent outside of a host cell under non-growing conditions is a potential stress condition indicating that the ability to communicate with the environment is likely critical to R. prowazekii pathogenesis. This R21 proposal will lay the foundation from which these extremely interesting questions can be further explored. Based on the prominent role of two-component response regulators in sensing environmental changes, I have selected the R. prowazekii ORFs RP426/RP427 (annotated as EnvZ/OmpR) as a potential sensor kinase/response regulator pair and will elucidate the regulon of genes under their control. The goal of this R21 proposal is to use this two-component network as a relevant biological model to adapt the Chromatin Immunoprecipitation (ChIP) technique for use on an obligate intracellular, cytosol-limited pathogen. The Specific Aim describes two approaches. I will use purified, recombinant OmpR, phosphorylated OmpR (OmpR approximately P) and R. prowazekii chromosomal DNA fragments to perform an in vitro ChIP assay. OmpR/OmpR approximately P-bound regulatory sequences will be isolated, cloned, and identified by DNA sequencing. This in vitro approach will identify regulatory sequences independent of environmental conditions. The second approach is an in vivo ChIP assay that will isolate regulatory DNA sequences from R. prowazekii grown under controlled environmental conditions. The conditions will be selected based on the data generated by the in vitro ChIP and also based on the limited number of environmental niches to which rickettsiae are exposed. The combination of these two techniques will elucidate R. prowazekii genes controlled by OmpR and could provide insight into the environmental stimuli to which this system responds. Consistent with the exploratory/development nature of an R21 application, this study represents the first effort to develop chromatin immunoprecipitation to elucidate a gene regulon in an obligate, intracellular pathogen using R. prowazekii as the model system. Understanding the role of gene regulation in pathogenesis may be critical to developing countermeasures and novel therapies against obligate intracellular Select Agents.