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）：立克属的成员是术内病原体，引起人类流行病和流行斑疹伤寒等疾病。据估计，流行病的虱子矢量特工R. Prowazekii在上个世纪造成了超过300万人死亡！ R. prowazekii是一种精选药物，已被NIAID指定为B类优先病原体。在体内，立克西亚仅限于有限数量的环境壁ni，包括在虱子载体的肠道上皮细胞中的生长到人类宿主的血液与人体内皮细胞之间的过渡。考虑到成功适应真核细胞质生长的生长生长生长的生物的稀少，可以将细胞质视为一种敌对的环境。此外，由于这种强制性细胞内生活方式的性质，在非生长条件下在宿主细胞外花费的任何时间都是潜在的应力条件，表明与环境通信的能力可能对R. prowazekii发病机理至关重要。该R21提案将奠定这些非常有趣的问题的基础。基于两个组分子响应调节剂在感应环境变化中的重要作用，我选择了R. prowazekii ORFS RP426/RP427（注释为ENVZ/OMPR）作为潜在的传感器激酶/响应调节器对，并将阐明其控制下的基因调节。该R21提案的目的是将此两组分网络用作相关的生物学模型，以适应染色质免疫沉淀（CHIP）技术，以用于强制性细胞内，细胞质含量有限的病原体。具体目的描述了两种方法。我将使用纯化的重组OMPR，磷酸化的OMPR（OMPR大约P）和R. prowazekii染色体DNA片段进行体外CHIP测定。 OMPR/OMPR将通过DNA测序分离，克隆和鉴定大约P-BON-BON结合调节序列。这种体外方法将确定与环境条件无关的调节序列。第二种方法是一种体内芯片测定法，该测定法可以将调节性DNA序列与受控环境条件下生长的R. prowazekii分离。条件将根据体外芯片生成的数据，以及基于Rickettsiae暴露到的环境壁ni的数量有限的数据。这两种技术的组合将阐明由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应用的探索性/发育性质一致，这项研究代表了开发染色质免疫沉淀的首次努力，以使用R. prowazekii作为模型系统阐明具有强制性的细胞内病原体中的基因调节。了解基因调节在发病机理中的作用对于开发对策和针对强制性细胞内选择药物的新疗法至关重要。