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利希菌病理生物学中的作用。查菲埃里希氏菌是一种专性胞内细菌，是人类单核细胞增多性埃立克体病(HME)的病原体，HME是一种新出现的威胁生命的人畜共患病。在查菲埃希氏菌感染过程中，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免疫反应、细胞凋亡、膜转运、信号转导和细胞周期的基因。查菲埃希氏菌gp200是本实验室鉴定的最大的埃利希菌糖蛋白，已在感染埃立克氏菌的单核细胞中被鉴定出来。虽然巨噬细胞的基因表达似乎发生了改变，有利于埃利希氏菌的存活，但埃利希氏核转位蛋白在基因调控中所起的作用尚不清楚。关于gp200在巨噬细胞核中的作用的科学知识的这种差距，阻碍了理解埃利希菌的病理生物学和操纵这些医学上重要的细菌。这项提议的目的是确定gp200核转位所涉及的机制，并确定gp200核目标。我们假设，查菲肠杆菌gp200是通过依赖于糖的核输入途径转移到细胞核的，在那里它作为转录抑制物、激活剂和/或调节器促进细胞内埃利西里细胞的存活。我们提出了以下具体目标：1)确定参与gp200转位到单核细胞的宿主细胞核输入途径；2)确定在感染过程中与gp200相互作用的单核细胞核蛋白和DNA基序。核进口途径将通过观察荧光gp200在多种途径抑制剂存在下的核转位来绘制。将进行蛋白质/DNA阵列和SELEX分析以鉴定与gp200结合的宿主基因组序列，并通过蛋白质/蛋白质阵列和免疫共沉淀鉴定gp200的核蛋白结合伙伴。综上所述，这些结果将加深我们对致病细菌糖蛋白功能的理解，并确定蛋白质糖基化在埃利希菌毒力中的作用。HME是一种新兴的公共卫生问题，这项研究将促进针对北美最普遍的威胁生命的壁虱传播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.