Ehrlichia T1S Effector Regulation of Host Gene Transcription

描述（由申请方提供）：查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隔室中，形成称为桑椹胚的膜结合小菌落。探讨了E. chaffeensis是内化，建立细胞内感染，并避免先天性宿主防御是不理解的，但似乎发生通过功能相关的宿主-病原体相互作用与新描述的1型分泌（T1 S）串联重复蛋白（TRP）效应。我们已经确定，TRP 120易位到宿主细胞核，包含一个新的TR DNA结合域，结合GC丰富的DNA基序，是宿主泛素（Ub）和小泛素样修饰（SUMO）翻译后修饰（PTM）途径的底物，这是已知的广泛扩展真核生物蛋白质的相互作用和功能能力。本研究的目的是证明TRP 120是一种双功能转录因子，与细胞凋亡和细胞凋亡之间存在相互作用。 Ub/SUMO途径介导核转位、亚核缔合和调节宿主防御基因表达。通过这项研究，我们将定义的调节机制介导的TRP 120运输到细胞核，表征亚核定位和相互作用，确定TRP 120-DNA结合的分子基础，以及TRP 120和PTMs在调节宿主基因表达的作用，使用最先进的分子和细胞方法。在第一个目标中，我们将使用TRP 120突变体和小分子抑制剂来确定真核细胞PTMs在TRP 120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，TRP 120-DNA相互作用，转录因子的功能，和TRP 120 PTM的作用将使用分子方法，包括单酵母杂交，体外转录，和哺乳动物报告基因测定进行研究。在目标三中，将使用TRP 120靶基因表达阵列和染色质免疫沉淀法来测定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，从而检查TRP 120在细胞背景中调节宿主靶基因表达和表观遗传模式的能力。随着微生物核效应子在病理生物学中的重要作用的出现，分子表征的埃里希体TRP效应子提供了一个相关的和明确定义的模型，用于研究通过病原体定向操纵吞噬细胞的分子策略直接转录调节宿主基因的机制。表征宿主细胞的埃里希体效应和分子机制，通过这些被介导的调制途径将扩大我们的细胞内微生物的感染和生存的细胞生物学的理解。这是必要的，以确定新的宿主靶标的治疗的基础上，机械定义的宿主-病原体相互作用可能利用各种各样的病原体。

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.