TYPE IV SECRETION & SIGNAL TRANSDUCTION IN EHRLICHIOSIS

描述（由申请人提供）：由沙菲埃立体引起的人单核细胞埃立体病（HME）和由嗜吞噬细胞无原体引起的人粒细胞埃立体病（HGE），均为新兴传染病。这些细菌是挑剔的强制性细胞内细菌，需要在两个截然不同的宿主（脊椎动物和无脊椎动物）之间反复传播。这些细菌如何进入并继续在中性粒细胞或蜱虫媒介等敌对宿主环境中茁壮成长在很大程度上是未知的。我们最近在HGE和HME药物中鉴定了编码IV型分泌系统（T4SS）和双组分调节系统（2CRS）的基因。本提案旨在了解T4SS和2CRS在HME和HGE发病机制中的作用。本项目的具体目的如下：1。在附着、内化和随后的增殖过程中，以及对环境刺激的反应中，研究沙菲沙芽胞杆菌和嗜吞噬胞杆菌个体表达的T4SS蛋白与人白细胞内溶酶体标记物和信号分子的共定位。2. 表征蜱传播过程中T4SS在蜱和哺乳动物皮肤中的表达。3. 鉴定T4SS效应分子或底物分子。4. 通过表达重组蛋白、研究传感器激酶和反应调节因子在体外和体内的磷酸化，在可控的环境条件下表征沙菲沙螨和嗜吞噬细胞沙螨的2CRS；研究组氨酸激酶（2CRS）抑制剂对沙菲棘球蚴和嗜吞噬细胞棘球蚴表型的影响；以及通过研究受2CRS调控的基因。5. 构建新的a突变体。其中一个预测的关键基因被破坏，或有条件地沉默以决定T4SS和2CRS的功能。本研究的数据将为理解细胞内细菌及其哺乳动物和蜱虫宿主之间的动态信号事件提供新的视角。这个项目有可能在这个研究领域带来技术突破。这一结果可能为HME和HGE的治疗和预防指明一个潜在的靶点。

DESCRIPTION (provided by the applicant): Human monocytic ehrlichiosis (HME) caused by Ehrlichia chaffeensis and human granulocytic ehrlichiosis (HGE) caused by Anaplasma phagocytophila, are emerging infectious diseases. These bacteria are fastidious obligatory intracellular bacteria that require repeated transmission between two vastly different hosts: vertebrates and invertebrates. How these bacteria enter and continue to thrive within hostile host milieu such as neutrophils or the tick vector is largely unknown. We recently characterized genes encoding a type IV secretion system (T4SS) and two component-regulatory system (2CRS) in HGE and HME agents. This proposal aims to understand the roles of T4SS and 2CRS in pathogenesis of HME and HGE. The specific aims of this project are as follows: 1. Examine the colocalization of T4SS proteins expressed by individual E. chaffeensis and A. phagocytophila organisms with endosome-lysosome markers and signaling molecules in human leukocytes during attachment, internalization, and subsequent proliferation, and in response to environmental stimuli. 2. Characterize expression of T4SS in ticks and the skin of mammals during tick transmission. 3. Identify T4SS effector or substrate molecules. 4. Characterize the 2CRS of E. chaffeensis and A. phagocytophila by expressing recombinant proteins and studying the phosphorylation of sensor kinases and response regulators in vitro, and in vivo under controlled sets of environmental conditions; by examining the effects of 2CRS (histidine kinase) inhibitors on phenotypes of E. chaffeensis and A. phagocytophila; and by studying genes regulated by 2CRS. 5. Construct novel mutants ofA. phagocytophila and E. chaffeensis in which a predicted critical gene is disrupted, or conditionally silenced to determine the functions of T4SS and 2CRS. The data obtained from this study will provide a new perspective for understandings the dynamic signaling events between obligatory intracellular bacteria and their mammalian and tick hosts. This project will potentially provide a technical breakthrough in this field of research. The results may point to a potential target for new treatment and prevention of HME and HGE.