TYPE IV SECRETION & SIGNAL TRANSDUCTION IN EHRLICHIOSIS

描述(由申请人提供)：由查菲埃立克体引起的人类单核细胞埃立克体病(HME)和由吞噬无浆体引起的人粒细胞埃立克体病(HGE)是新出现的传染病。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之间重复传播：脊椎动物和无脊椎动物。这些细菌是如何进入并继续在中性粒细胞或扁虱媒介等敌意宿主环境中茁壮成长的，在很大程度上是未知的。我们最近在HGE和HME制剂中鉴定了编码一个IV型分泌系统(T4SS)和两个组分调节系统(2CRS)的基因。本研究旨在了解T4SS和2CRS在HME和HGE发病机制中的作用。本项目的具体目标如下：1.研究查菲埃希氏菌和吞噬细胞性艾美耳球虫表达的T4SS蛋白在人白细胞附着、内化和随后的增殖过程中，以及对环境刺激的反应中，与内体溶酶体标记物和信号分子的共存。2.研究T4SS在蜱类和哺乳动物皮肤中的表达。3.鉴定T4SS效应子或底物分子。4.在一定的环境条件下，通过表达重组蛋白、研究感受蛋白和反应调节因子的磷酸化，通过检测2CRS抑制剂对查菲埃希氏菌和吞噬艾美耳球虫表型的影响，以及研究2CRS对2CRS的调控基因，研究2CRS对查菲埃希氏菌和吞噬艾美耳球虫表型的影响。5.构建新的突变体OFA。吞噬细胞和查菲埃希氏菌，其中一个预测的关键基因被打乱，或有条件地沉默，以确定T4SS和2CRS的功能。这项研究的数据将为理解必需的细胞内细菌与它们的哺乳动物和壁虱宿主之间的动态信号事件提供一个新的视角。这一项目可能会在这一研究领域提供技术突破。这一结果可能为HME和HGE的新治疗和预防提供一个潜在的靶点。

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.