Elucidating the Rickettsia prowazekii EnvZ/OmpR Regulon

阐明普瓦泽基立克次体 EnvZ/OmpR 调节子

• 批准号: 7286854
• 负责人: JONATHON PETER AUDIA
• 金额: $ 17.83万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-15 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7286854
• 关键词: Animal Model; Antibodies; Arthropod Vectors; Arthropods; Bacteria; Binding; Binding Sites; Biological; Biological Assay; Biological Models; Biology; Blood; Categories; Cell Line; Cells; Cessation of life; Chromatin; Cloning; Collection; Complex; Condition; Cosmids; Cytolysis; Cytosol; DNA; DNA Sequence; DNA-Directed RNA Polymerase; Data; Development; Disease; Electrophoretic Mobility Shift Assay; Endothelial Cells; Environment; Epidemic; Epithelial Cells; Escherichia; Escherichia coli; Family; Fibroblasts; Formaldehyde; Foundations; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genome; Goals; Growth; Human; Immunoprecipitation; In Vitro; Infection; Lead; Lice; Life Style; Ligation; Longitudinal Studies; Mediating; Mus; National Institute of Allergy and Infectious Disease; Nature; Numbers; OmpR protein; Open Reading Frames; Organism; Orthologous Gene; Pathogenesis; Phosphotransferases; Plasmids; Polymerase Chain Reaction; Recombinants; Regulon; Rewards; Rickettsia; Rickettsia prowazekii; Risk; Role; Signal Transduction; Staging; Stimulus; Stream; Stress; Superhelical DNA; System; Techniques; Testing; Thinking; Time; Typhus; base; chromatin immunoprecipitation; crosslink; environmental change; genetic regulatory protein; in vivo; insight; interest; macrophage; member; mutant; novel; pathogen; response; sensor; success; vector

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.

描述（由申请人提供）：立克次体属成员是专性胞浆内病原体，可引起人类流行性和地方性斑疹伤寒等疾病。据估计，由虱子传播的流行性斑疹伤寒病原体普瓦兹基氏菌 (R. prowazekii) 在上个世纪已导致超过 300 万人死亡！ R. prowazekii 是一种精选病原体，已被 NIAID 指定为 B 类优先病原体。在体内，立克次体被限制在有限数量的环境生态位中，包括在虱子载体的肠上皮细胞中的生长到人类宿主的血流和人类内皮细胞之间的转变。考虑到成功适应真核细胞质作为生长生态位的生物体很少，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，表明与环境沟通的能力可能对普罗瓦泽基菌发病机制至关重要。 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突出作用，我选择了R. prowazekii ORFs RP426/RP427（注释为EnvZ/OmpR）作为潜在的传感器激酶/反应调节因子对，并将阐明基因的调节在他们的控制之下。 R21 提案的目标是使用这种两部分网络作为相关的生物学模型，以适应染色质免疫沉淀 (ChIP) 技术，用于专性细胞内、胞质限制的病原体。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 片段来进行体外 ChIP 测定。 OmpR/OmpR 大约 P 结合调节序列将被分离、克隆并通过 DNA 测序进行鉴定。这种体外方法将识别独立于环境条件的调控序列。第二种方法是体内 ChIP 测定，它将从在受控环境条件下生长的 R. prowazekii 中分离出调控 DNA 序列。将根据体外 ChIP 生成的数据以及立克次体暴露的环境生态位的有限数量来选择条件。这两种技术的结合将阐明 OmpR 控制的 R. prowazekii 基因，并可以深入了解该系统响应的环境刺激。与 R21 应用的探索/开发性质一致，本研究代表了首次尝试开发染色质免疫沉淀，以使用 R. prowazekii 作为模型系统来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对于开发针对专性细胞内选择剂的对策和新疗法至关重要。