Ehrlichia Type-1 Secreted Effectors Exploit Host SUMO Pathways

描述(申请人提供)：查菲埃立克体病是人类单核细胞增生性埃立克体病的病原体，这是美国最普遍的威胁生命的人畜共患病。查菲埃希氏菌是一种专性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埃利西尔效应蛋白的未知机制逃避免疫反应。为了进一步了解查菲埃希氏菌的病理生物学，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，查菲埃希氏菌利用1型分泌系统(T1S)输出串联重复蛋白(TRP)效应器，该效应器与宿主蛋白的功能不同的网络相互作用，通过该网络调节宿主细胞的功能。然而，这些相互作用的潜在分子基础和介导的途径尚未定义。在这里，我们证明了查菲埃希氏菌T1S效应器TRP120通过宿主途径被小泛素样修饰物(SUMO)修饰。SUMO修饰介导高亲和力的蛋白质与宿主蛋白的相互作用，包含一个共同的相扑相互作用基序(SIM)，人们正在意识到这种修饰所诱导的各种细胞功能。真核生物利用相扑翻译后修饰来动态扩展蛋白质的功能，并介导蛋白质之间的相互作用、亚细胞定位、基因表达和蛋白质降解。众所周知，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的一种手段，以促进感染。我们的发现是Ehrlichial TRPs是宿主SUMO化途径的底物，这是首次描述了通过这一途径对原核蛋白进行直接修饰。我们建议将Ehrlichial T1S效应器直接相联，作为一种实现宿主蛋白相互作用、动态调节效应器亚细胞定位以及调节支持细胞内感染和生存的宿主细胞功能的手段。这些研究的长期目标是确定T1S效应器在血液病理生物学中的作用，以及PTM在效应器功能中的作用。这项建议的目的是证明埃利西尔色氨酸效应器通过宿主PTM途径的相扑结合，这是与促进感染和细胞内生存的不同宿主蛋白相互作用的分子基础。在目标1中，我们将证明Ehrlichial TRPs是直接SUMO共轭的，并从分子上定义了这种修饰的基础。这些研究将为AIM 2提供必要的工具，在该研究中，我们建议在查菲埃希氏杆菌感染的背景下评估效应器SUMO化的功能影响。总之，本研究将表征这些效应器和PTM在埃利希菌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洞察力。

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.