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易位蛋白在卵泡细胞病理生物学中的作用。沙菲埃立体是一种专性细胞内细菌，是人类嗜单核细胞埃里希体病（HME）的病原体，HME是一种危及生命的新发蜱传人畜共患病。在沙非叶虫感染期间，许多单核细胞基因的转录水平发生改变，包括那些参与先天和细胞介导的免疫应答、凋亡、膜运输、信号转导和细胞周期的基因。在我们实验室鉴定的最大的埃利希体糖蛋白沙菲伊夫gp200已经在埃利希体感染单核细胞的细胞核内被鉴定出来。尽管巨噬细胞的基因表达似乎被改变以有利于埃氏细胞的存活，但埃氏细胞来源的核易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差距是理解细胞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gp200核易位的机制，并确定gp200核靶点。我们假设沙菲沙蚕gp200通过聚糖依赖的核输入途径易位到细胞核，在那里它作为转录抑制因子、激活因子和/或调节剂促进细胞内卵泡细胞存活。我们提出以下具体目标：1)确定gp200易位到单核细胞核的宿主细胞核输入途径；2)确定感染过程中与gp200相互作用的单核细胞核蛋白和DNA基序。核输入途径将通过观察荧光gp200在多种途径抑制剂存在下的核易位来绘制。通过蛋白/DNA阵列和SELEX分析鉴定gp200结合的宿主基因组序列，通过蛋白/蛋白阵列和共免疫沉淀鉴定gp200的核蛋白结合伙伴。总之，这些结果将进一步加深我们对病原菌糖蛋白功能的理解，并确定蛋白质糖基化在埃氏体毒力中的作用。HME是一个新兴的公共卫生问题，这项研究将促进开发新的治疗干预措施，以对抗北美最普遍的威胁生命的蜱传疾病之一。

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.