Ehrlichia Glycoprotein Modulation of Macrophage Gene Expression

描述（由申请人提供）：本研究项目的长期目标是了解核转位蛋白在埃里希体病理生物学中的作用。查菲埃里希体是一种专性胞内细菌，是人类嗜单核细胞埃里希体病（HME）的病原体，HME是一种威胁生命的新型蜱传人畜共患病。在E.在Chaffeensis感染后，许多单核细胞基因的转录水平发生改变，包括参与先天性和细胞介导的免疫应答、细胞凋亡、膜运输、信号转导和细胞周期的那些。急诊Chaffeensis gp 200是我们实验室鉴定的最大埃立克体糖蛋白，已在埃立克体感染的单核细胞的细胞核内鉴定。虽然巨噬细胞基因表达的改变似乎有利于埃里希体的生存，埃里希体起源的核转位蛋白在基因调控中的作用是未知的。关于gp 200在巨噬细胞核中的作用的科学知识的这一空白是理解埃利希病理生物学和操纵这些医学上重要的细菌的障碍。本提案的目的是确定参与gp 200核转位的机制，并确定gp 200核靶点。我们假设E. ChaffeensisGP 200通过聚糖依赖性核输入途径转移到细胞核，在那里它作为转录抑制物、激活物和/或调节物促进细胞内埃里希体存活。我们提出以下具体目标：1）确定参与gp 200易位到单核细胞核的宿主细胞核输入途径，和2）鉴定在感染期间与gp 200相互作用的单核细胞核蛋白和DNA基序。通过观察在多种途径抑制剂存在下荧光gp 200的核转位来绘制核输入途径。将进行蛋白质/DNA阵列和SELEX分析以鉴定gp 200结合的宿主基因组序列，并将使用蛋白质/蛋白质阵列和免疫共沉淀鉴定gp 200的核蛋白结合配偶体。总之，这些结果将进一步了解致病性细菌糖蛋白的功能，并确定蛋白质糖基化在埃立克体毒力的作用。HME是一个新兴的公共卫生问题，这项研究将促进针对北美最流行的威胁生命的蜱传疾病之一的新治疗干预措施的开发。

DESCRIPTION (provided by applicant): The long term goal of this research project is to understand the role of nuclear translocated proteins in ehrlichial pathobiology. Ehrlichia chaffeensis, an obligate intracellular bacterium, is the agent of human monocytotropic ehrlichiosis (HME), a life-threatening emerging tick-borne zoonosis. During E. chaffeensis infection, transcription levels of numerous monocyte genes are altered including those involved in innate and cell-mediated immune responses, apoptosis, membrane trafficking, signal transduction and cell cycle. The E. chaffeensis gp200, the largest ehrlichial glycoprotein characterized in our laboratory, has been identified within the nuclei of Ehrlichia-infected monocytes. Although macrophage gene expression appears to be altered to favor ehrlichial survival, the role played in gene regulation by nuclear translocated proteins of ehrlichial origin is unknown. This gap in scientific knowledge regarding the role of gp200 in the macrophage nucleus is an impediment towards understanding ehrlichial pathobiology and manipulating these medically important bacteria. The objective of this proposal is to determine the mechanism involved in gp200 nuclear translocation and to identify gp200 nuclear targets. We hypothesize that E. chaffeensis gp200 is translocated to the nucleus via a glycan-dependant nuclear import pathway, where it acts as a transcriptional repressor, activator and/or regulator to promote intracellular ehrlichial survival. We propose the following specific aims: 1) determine the host cell nuclear import pathway involved in the translocation of gp200 to the monocyte nucleus, and 2) identify the monocyte nuclear proteins and DNA motifs that interact with gp200 during infection. The nuclear import pathway will be mapped by observing the nuclear translocation of fluorescent gp200 in the presence of numerous pathway inhibitors. A protein/DNA array and SELEX analysis will be performed to identify the host genomic sequences bound by gp200, and the nuclear protein binding partners of gp200 will be identified with a protein/protein array and co- immunoprecipitation. Together, these results will further our understanding of pathogenic bacterial glycoprotein function and identify a role for protein glycosylation in ehrlichial virulence. HME is an emerging public health concern, and this research will facilitate development of new therapeutic interventions against one of the most prevalent life-threatening tick-borne diseases in North America.