Ehrlichia T1S Effector Regulation of Host Gene Transcription

埃里希体 T1S 宿主基因转录的效应调节

• 批准号: 8824870
• 负责人: JERE W MCBRIDE
• 金额: $ 38.37万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8824870
• 关键词: Acute Promyelocytic Leukemia; Amino Acids; Apoptosis; Bacteria; Bacterial Infections; Binding; Biological Assay; Body Composition; C-terminal; Cell Cycle; Cell Cycle Regulation; Cell Nucleus; Cell physiology; Cells; Cellular biology; Chromatin; DNA; DNA Binding; DNA Binding Domain; DNA Modification Process; Data; Defect; Defense Mechanisms; Development; Early Endosome; Ehrlichia; Ehrlichia chaffeensis; Ehrlichiosis; Epigenetic Process; Eukaryota; Gene Activation; Gene Expression; Genes; Genetic Transcription; Global Change; Goals; Health; Host Defense; Human; Hybrids; Immune; Immune response; In Vitro; Infection; Invaded; Investigation; Life; Mediating; Membrane; Microbe; Modeling; Modification; Molecular; Mononuclear; Monoubiquitination; Morula; Nuclear; Nuclear Import; Nuclear Translocation; Organism; Pathway interactions; Pattern; Phagocytes; Post-Translational Protein Processing; Process; Prokaryotic Cells; Property; Proteins; Regulation; Reporter Genes; Reporting; Research; Role; Signal Transduction; Site; Specificity; Tandem Repeat Sequences; Therapeutic; Ticks; Transactivation; Transcriptional Activation; Transcriptional Regulation; Ubiquitin; Yeasts; Zoonoses; adaptive immunity; antimicrobial; base; biological adaptation to stress; cellular targeting; chromatin immunoprecipitation; gene repression; histone modification; human disease; inhibitor/antagonist; insight; microbial; mutant; nanomachine; novel; pathogen; prevent; protein complex; small molecule; therapeutic target; trafficking; transcription factor

DESCRIPTION (provided by applicant): Ehrlichia chaffeensis selectively infects mononuclear phagocytes and resides in early-endosome-like compartments, forming membrane-bound microcolonies called morulae. The mechanisms by which E. chaffeensis is internalized, establishes intracellular infection and avoids innate host defenses are not understood, but appear to occur through functionally relevant host-pathogen interactions associated with newly described type 1 secretion (T1S) tandem repeat protein (TRP) effectors. We have determined that TRP120 is translocated into the host cell nucleus, contains a novel TR DNA binding domain, binds a GC-rich DNA motif, and is a substrate of host ubiquitin (Ub) and small ubiquitin-like modifier (SUMO) post translational modification (PTM) pathways, which are known to extensively expand interactive and functional capability of eukaryotic proteins. The goal of this study is to demonstrate that TRP120 is a dual-function transcription factor that has interplay with the Ub/SUMO pathways to mediate nuclear translocation, subnuclear associations and regulate host defense gene expression. Through this investigation we will define the regulatory mechanisms mediating TRP120 trafficking to the nucleus, characterize subnuclear localization and interactions, determine the molecular basis of TRP120-DNA binding, and role of TRP120 and PTMs in modulating host gene expression using state-of-the-art molecular and cellular approaches. In the first aim, we will use TRP120 mutants and small molecule inhibitors to determine the role of eukaroytic PTMs in TRP120 nuclear localization and define subnuclear localization and molecular interactions. In the second aim, TRP120-DNA interactions, transcription factor function, and role of TRP120 PTMs will be investigated using molecular approaches including one-yeast hybrid, in vitro transcription, and mammalian reporter gene assays. The capacity of TRP120 to modulate host target gene expression and epigenetic patterns in a cellular context will be examined in aim three using TRP120 target gene expression arrays and chromatin immunoprecipitation to determine DNA and histone modifications as well as functional assays to examine defects in cellular defense mechanisms. As the important role of microbial nuclear effectors in pathobiology is emerging, the molecularly characterized Ehrlichia TRP effectors offer a relevant and well defined model for investigating mechanisms of direct transcriptional modulation of host genes by this molecular strategy of pathogen-directed manipulation of the phagocyte. Characterization of the host cell pathways modulated by ehrlichial effectors and the molecular mechanisms through which these are mediated will expand our understanding of the cell biology of infection and survival by intracellular microbes. This is necessary to identify novel host targets for therapeutics based on mechanistically defined host-pathogen interactions potentially utilized by a wide variety of pathogens.

描述（由申请人提供）：恰菲埃里希体选择性感染单核吞噬细胞并驻留在早期内体样区室中，形成称为桑葚胚的膜结合微菌落。查菲埃里希体内化、建立细胞内感染和避免先天宿主防御的机制尚不清楚，但似乎是通过与新描述的 1 型分泌 (T1S) 串联重复蛋白 (TRP) 效应子相关的功能相关的宿主-病原体相互作用而发生的。我们已经确定 TRP120 易位到宿主细胞核中，包含一个新的 TR DNA 结合结构域，结合富含 GC 的 DNA 基序，并且是宿主泛素 (Ub) 和小泛素样修饰剂 (SUMO) 翻译后的底物修饰（PTM）途径，已知可广泛扩展真核蛋白质的相互作用和功能能力。本研究的目的是证明 TRP120 是一种双功能转录因子，与 Ub/SUMO 途径介导核易位、亚核关联并调节宿主防御基因表达。通过这项研究，我们将确定介导 TRP120 运输到细胞核的调节机制，表征亚核定位和相互作用，确定 TRP120-DNA 结合的分子基础，以及 TRP120 和 PTM 在使用最新技术调节宿主基因表达中的作用。艺术分子和细胞方法。第一个目标是，我们将使用 TRP120 突变体和小分子抑制剂来确定真核 PTM 在 TRP120 核定位中的作用，并定义亚核定位和分子相互作用。在第二个目标中，将使用分子方法（包括单酵母杂交、体外转录和哺乳动物报告基因测定）研究 TRP120-DNA 相互作用、转录因子功能和 TRP120 PTM 的作用。 TRP120 在细胞环境中调节宿主靶基因表达和表观遗传模式的能力将在目标三中进行检查，使用 TRP120 靶基因表达阵列和染色质免疫沉淀来确定 DNA 和组蛋白修饰以及功能测定来检查细胞防御机制中的缺陷。随着微生物核效应子在病理学中的重要作用逐渐显现，分子特征埃里希体 TRP 效应子提供了一个相关且明确的模型，用于通过这种病原体定向操纵吞噬细胞的分子策略来研究宿主基因的直接转录调节机制。埃利希体效应子调节的宿主细胞途径的表征以及介导这些途径的分子机制将扩大我们对细胞内微生物感染和存活的细胞生物学的理解。这对于根据多种病原体可能利用的机械定义的宿主-病原体相互作用来确定新的宿主治疗靶标是必要的。