Elucidating the Rickettsia prowazekii EnvZ/OmpR Regulon

描述（由申请人提供）：立克次体属成员是专性胞质内病原体，可引起人类流行性和地方性斑疹伤寒等疾病。估计R.在上个世纪，传染性斑疹伤寒的虱媒prowazekii已经造成了300多万人死亡！R. prowazekii是一种选择性病原体，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小生境，包括在虱载体的肠上皮细胞中生长到人类宿主的血流和人类内皮细胞之间的转变。考虑到已经成功地适应在真核细胞质中生长的生物体的缺乏，细胞质可以被认为是一个敌对的环境。此外，由于这种专性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潜在的应激条件，表明与环境交流的能力可能对R至关重要。prowazekii发病机制。这一R21建议将为进一步探讨这些极其有趣的问题奠定基础。基于双组分响应调节因子在感知环境变化中的突出作用，我选择了R。prowazekii ORF RP 426/RP 427（注释为EnvZ/OmpR）作为潜在的传感器激酶/反应调节子对，并将阐明在其控制下的基因的调节子。这个R21提案的目标是使用这种双组分网络作为相关的生物模型，以适应染色质免疫沉淀（ChIP）技术用于专性细胞内，胞质限制性病原体。具体目标描述了两种方法。我将使用纯化的重组OmpR、磷酸化OmpR（OmpR约为P）和R。prowazekii染色体DNA片段进行体外ChIP测定。将通过DNA测序分离、克隆和鉴定OmpR/OmpR近似P结合的调控序列。这种体外方法将鉴定不依赖于环境条件的调控序列。第二种方法是体内ChIP测定，其将从R.在受控环境条件下生长的prowazekii。将根据体外ChIP生成的数据以及立克次体暴露的有限数量的环境小生境选择条件。这两种技术的结合将阐明R. prowazekii基因由OmpR控制，可以提供对该系统响应的环境刺激的洞察。与R21应用的探索性/开发性质一致，本研究代表了使用R. prowazekii作为模型系统。了解基因调控在发病机制中的作用可能对开发针对专性细胞内选择剂的对策和新疗法至关重要。

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.