Ehrlichia T1S Effector Regulation of Host Gene Transcription

描述(申请人提供)：查菲埃利希菌选择性地感染单核吞噬细胞，并居住在早期内吞体样隔室，形成膜结合的微集落，称为桑椹。查菲肠杆菌内化、建立细胞内感染和避免固有宿主防御的机制尚不清楚，但似乎是通过与新描述的类型1分泌(T1S)串联重复蛋白(Trp)效应器相关的功能相关的宿主-病原体相互作用发生的。我们已经确定TRP120被转位到宿主细胞核中，含有一个新的TRDNA结合域，结合了一个富含GC的DNA基序，是宿主泛素(Ub)和小泛素样修饰物(SUMO)翻译后修饰(PTM)途径的底物，这两条途径被认为可以广泛扩大真核蛋白质的相互作用和功能。这项研究的目的是证明TRP120是一个双功能转录因子，它与 Ub/SUMO途径介导核转位、亚核联合和调节寄主防御基因的表达。通过这项研究，我们将确定介导TRP120转运到细胞核的调控机制，表征核下定位和相互作用，确定TRP120与DNA结合的分子基础，以及TRP120和PTMS在利用最先进的分子和细胞方法调控宿主基因表达中的作用。在第一个目标中，我们将使用TRP120突变体和小分子抑制剂来确定真核裂解PTMS在TRP120核定位中的作用，并确定亚核定位和分子相互作用。在第二个目标中，将使用包括单酵母杂交、体外转录和哺乳动物报告基因分析在内的分子方法来研究TRP120与DNA的相互作用、转录因子功能和TRP120 PTM的作用。在目标三中，将使用TRP120靶基因表达阵列和染色质免疫沉淀来确定DNA和组蛋白的修饰，以及使用功能分析来检查细胞防御机制中的缺陷，来研究TRP120在细胞环境中调节宿主目标基因表达和表观遗传模式的能力。随着微生物核效应分子在病理生物学中的重要作用的出现，埃利希菌Trp效应分子的分子特征为研究病原体直接操纵吞噬细胞的分子策略对宿主基因的直接转录调控机制提供了一个相关的和明确的模型。对埃利希体效应器调控的宿主细胞通路及其介导的分子机制的表征将扩大我们对细胞内微生物感染和生存的细胞生物学的理解。这对于根据各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治疗学的新宿主靶点是必要的。

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.