Molecular basis of Wnt activation by Ehrlichia Wnt ligand mimics

项目摘要 查菲埃立克体（Ehrlichia chaffeensis）; ch.）是一种革兰氏阴性专性胞内细菌，是 在美国最流行的威胁生命的蜱传疾病，人单核细胞埃立克体病（HME）。 Wnt信号转导是一种保守的真核生物信号级联，包括经典和非经典途径， 调节事件，包括细胞命运，发育和细胞极性，以及先天免疫相关 例如自噬、细胞因子表达和吞噬作用。我们的实验室表明， 感染、E. ch.激活保守的真核细胞信号通路，包括经典和非经典信号通路 Wnt信号。Wnt信号转导增强E. ch.通过驱动细菌摄取和抑制 埃里希体复制泡与溶酶体的融合。尽管这些研究已经确定了Wnt 途径激活作为E. ch.，识别所观察到的激活事件 现象仍然是知识方面的一个关键差距。在正常生理条件下，Wnt信号传导- 依赖性吞噬作用通过Wnt配体与10种卷曲蛋白（Fzd）之一的结合而启动。我们 初步数据表明，E. ch.表面蛋白TRP 120直接结合Fzd，具有同源性， 与保守的Wnt蛋白家族，并能刺激Wnt转录因子β- 连环蛋白。我们还发现，抑制Wnt信号传导阻断了埃里希体的进入，表明E。C.有效 通过激活Wnt依赖性吞噬作用建立感染。该项目的长期目标是 利用E. ch.操纵单核细胞Wnt信号传导作为模型来研究 在人类细胞内细菌感染期间利用Wnt信号传导。这项建议的目的是 定义了大肠杆菌的细菌配体和真核受体决定簇。ch.效应子驱动的Wnt激活 信号传导和进入单核细胞。我们假设埃里希体TRP是Wnt配体模拟物， 通过Wnt途径受体-辅助受体对激活经典和非经典Wnt信号传导， 增强细菌宿主细胞进入和细胞内存活。在具体目标1中，我们将研究埃利希尔TRP 经典和非经典Wnt信号传导的Wnt配体模拟激活。在具体目标2中，我们将定义 Wnt途径受体和辅助受体在Ehrlichial TRP驱动的Wnt信号激活中的作用 感染这项研究将提供深入了解进化保守的真核生物途径， 已经进化为用于细胞侵入和细胞内生长。我们确定一个水平的方法， Wnt信号可以被细胞内病原体劫持，这将为先前观察到的 现象以及潜在的抗菌治疗靶点

Project Summary Ehrlichia chaffeensis (E. ch.) is a gram-negative, obligately intracellular bacterium and causative agent of the most prevalent life-threatening tick-borne disease in the United States, human monocytic ehrlichiosis (HME). Wnt signaling is a conserved eukaryotic signal cascade comprising canonical and noncanonical pathways that regulate events including cell fate, development, and cell polarity, as well as innate immunity-associated events such as autophagy, cytokine expression, and phagocytosis. Our laboratory has shown that during infection, E. ch. activates conserved eukaryotic signaling pathways including both canonical and noncanonical Wnt signaling. Wnt signaling enhances E. ch. intracellular survival by driving bacterial uptake and inhibiting fusion of the ehrlichial replicative vacuole with the lysosome. Although these studies have identified Wnt pathway activation as a virulence strategy for E. ch., identification of an activating event for the observed phenomena remains a critical gap in knowledge. Under normal physiological conditions, Wnt signaling- dependent phagocytosis is initiated through the binding of a Wnt ligand to one of 10 Frizzled (Fzd). Our preliminary data demonstrates that E. ch. surface protein TRP120 directly binds a Fzd, possesses homology with the conserved family of Wnt proteins, and can stimulate activation of the Wnt transcription factor β- catenin. We have also shown that inhibition of Wnt signaling blocks ehrlichial entry, indicating E. ch. effectively establishes infection through activation of Wnt-dependent phagocytosis. The long-term goal of this project is to utilize E. ch. manipulation of monocyte Wnt signaling as a model to study the therapeutic potential of harnessing Wnt signaling during human intracellular bacterial infection. The objective of this proposal is to define the bacterial ligand and eukaryotic receptor determinants of E. ch. effector-driven activation of Wnt signaling and entry into monocytes. We hypothesize that ehrlichial TRPs are Wnt ligand mimetics that signal through Wnt pathway receptor-coreceptor pairs for activation of canonical and noncanonical Wnt signaling to enhance bacterial host cell entry and intracellular survival. In specific aim 1, we will investigate ehrlichial TRP Wnt ligand mimetic activation of canonical and noncanonical Wnt signaling. In specific aim 2, we will define the role of Wnt pathway receptors and coreceptors in ehrlichial TRP-driven Wnt signaling activation during infection. This research will provide insight to evolutionarily conserved eukaryotic pathways that pathogens have evolved to utilize for cell invasion and intracellular growth. Our approach to identifying a level at which Wnt signaling can be hijacked by intracellular pathogens will provide mechanisms for previously observed phenomena as well as potential antimicrobial therapeutic targets