Ehrlichia Type-1 Secreted Effectors Exploit Host SUMO Pathways

描述（由申请人提供）：沙菲埃立体是人类嗜单核细胞埃里希体病的病原体，是美国最常见的威胁生命的蜱传人畜共患病。沙菲奈瑟菌是一种专性细胞内革兰氏阴性细菌，它选择性地感染单核吞噬细胞，并通过依赖于分泌的卵泡效应蛋白的未明确机制逃避免疫反应。为了进一步了解沙非叶蝉的病理生物学，我们的研究重点是了解这些效应物在介导和建立细胞内感染中的作用。我们最近报道了沙菲沙蚕利用1型分泌（T1S）系统输出串联重复蛋白（TRP）效应物，这些效应物与功能多样的宿主蛋白网络相互作用，通过宿主蛋白网络调节宿主细胞功能。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。在此，我们证明了沙菲沙叶T1S效应物TRP120通过宿主途径被小泛素样修饰物（SUMO）修饰。sumo修饰介导了与宿主蛋白的高亲和力蛋白相互作用，宿主蛋白包含一个共同的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，介导蛋白质相互作用、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可以调节宿主PTM途径，作为劫持宿主细胞功能以促进感染的手段。我们发现埃氏体TRPs是宿主SUMOylation途径的底物，这是第一次描述通过该途径直接修饰原核蛋白。我们建议将胚状T1S效应物直接与sumo偶联，作为宿主蛋白相互作用、效应物亚细胞定位的动态调节以及支持细胞内感染和存活的宿主细胞功能的调节手段。这些研究的长期目标是表征T1S效应物在细胞病理生物学中的作用，以及PTMs在效应物功能中的作用。本研究的目的是通过宿主PTM途径证明卵泡型TRP效应物的sumo偶联，这是与多种宿主蛋白相互作用的分子基础，促进了感染和细胞内存活。在目标1中，我们将证明卵细胞TRPs是直接sumo共轭的，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，我们建议评估效应体SUMOylation在沙非叶蝉感染背景下的功能影响。总之，本研究将描述这些效应物和ptm在埃利希体病理生物学中的作用，并为细胞内微生物调节宿主细胞功能和建立感染的新机制提供见解。

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.