Ehrlichia Type-1 Secreted Effectors Exploit Host SUMO Pathways

描述（由申请方提供）：查菲埃里希体是人嗜单核细胞埃里希体病的病原体，这是美国最常见的危及生命的蜱传人畜共患病。E. Chaffeensis是一种专性细胞内革兰氏阴性细菌，通过依赖于分泌的埃里希体效应蛋白的未确定的机制选择性感染单核吞噬细胞并逃避免疫应答。为了进一步了解E.我们研究的重点是了解这些效应子在介导和建立细胞内感染中的作用。我们最近报道了E. Chaffeensis利用1型分泌（T1 S）系统输出串联重复蛋白（TRP）效应物，其与宿主蛋白质的功能多样性网络相互作用，通过其调节宿主细胞功能。然而，这些相互作用的潜在分子基础和介导的途径尚未确定。在这里，我们证明了E. Chaffeensis T1 S效应子TRP 120通过宿主途径被小泛素样修饰物（SUMO）修饰。SUMO化介导与宿主蛋白质的高亲和力蛋白质-蛋白质相互作用，包含共同的SUMO相互作用基序（SIM），并且对这种修饰诱导的各种细胞功能有了新的认识。真核生物利用SUMO翻译后修饰来动态扩展蛋白质功能并介导蛋白质-蛋白质相互作用、亚细胞定位、基因表达和蛋白质降解。已知病毒和细胞内细菌效应物调节宿主PTM途径作为劫持宿主细胞功能以促进感染的手段。我们发现埃里希体TRP是宿主SUMO化途径的底物，这代表了该途径对原核蛋白直接修饰的首次描述。我们提出，Ehrlichial T1 S效应子是直接SUMO缀合的，作为使宿主蛋白相互作用、动态调节效应子亚细胞定位和调节支持细胞内感染和存活的宿主细胞功能的手段。这些研究的长期目标是表征T1 S效应子在埃里希体病理学中的作用和PTM在效应子功能中的作用。本提案的目的是通过宿主PTM途径证明埃里希体TRP效应物的SUMO缀合，其作为与促进感染和细胞内存活的多种宿主蛋白相互作用的分子基础。在目标1中，我们将证明埃里希体TRP是直接SUMO结合的，并从分子上定义这种修饰的基础。这些研究将为目标2提供必要的工具，在目标2中，我们建议评估效应子SUMO化在大肠杆菌中的功能影响。查菲线虫感染总之，这项研究将表征这些效应子和PTM在埃里希体病理生物学中的作用，并深入了解细胞内微生物用于调节宿主细胞功能和建立感染的新机制。

DESCRIPTION (provided by applicant): Ehrlichia chaffeensis is the causative agent of human monocytotropic ehrlichiosis, the most prevalent life-threatening tick-borne zoonotic disease in the United States. E. chaffeensis, an obligately intracellular gram negative bacterium, selectively infects mononuclear phagocytes and evades immune response through undefined mechanisms that are dependent on secreted ehrlichial effector proteins. To further understand the pathobiology of E. chaffeensis the focus of our research is to understand the role of these effectors in mediating and establishing intracellular infection. We recently reported that E. chaffeensis utilizes type 1 secretion (T1S) system to export tandem repeat protein (TRP) effectors that interact with functionally diverse networks of host proteins, through which host cellular functions are modulated. However, the underlying molecular basis for these interactions and the pathways mediated are not defined. Herein, we demonstrate that the E. chaffeensis T1S effector, TRP120, is modified with the small ubiquitin-like modifier (SUMO) by the host pathway. SUMOylation mediates high affinity protein-protein interactions with host proteins encompassing a common SUMO-interacting motif (SIM), and there is an emerging appreciation for the varied cellular functions this modification induces. Eukaryotes utilize SUMO post translational modification to dynamically extend protein function and mediate protein-protein interactions, subcellular localization, gene expression, and protein degradation. Viral and intracellular bacteria effectors are known to modulate host PTM pathways as a means of hijacking host cellular functions to facilitate infection. Our finding that ehrlichial TRPs are substrates of host SUMOylation pathways represents the first description of direct modification of prokaryotic protein by this pathway. We propose ehrlichial T1S effectors are directly SUMO-conjugated as a means of enabling host protein interactions, dynamic regulation of effector subcellular localization, and modulation of host cell functions that support intracellular infectio and survival. The long-term goal of these studies is to characterize the role of T1S effectors in ehrlichial pathobiology and the role of PTMs in effector function. The objective of this proposal i to demonstrate SUMO-conjugation of ehrlichial TRP effectors by host PTM pathways, which serves as the molecular basis for interactions with diverse host protein that facilitate infection and intracellular survival. In aim 1 we will demonstrate ehrlichial TRPs are directly SUMOconjugated, and molecularly-define the basis for this modification. These studies will provide the tools necessary for aim 2, in which we propose to evaluate the functional impact of effector SUMOylation in the context of E. chaffeensis infection. In total, this research will characterize the roles of these effectors and PTMs in the pathobiology of Ehrlichia, and provide insight into a novel mechanism utilized by intracellular microbes to modulate host cell functions and establish infection.