Keys to prevent iron hijacking and infection by intracellular bacteria

革兰氏阴性专性细胞内细菌恰菲埃里希体 (Ech) 感染单核细胞 - 巨噬细胞，并引起新出现的蜱传人畜共患人类单核埃利希体病 (HME)。我们的长期 长期目标是确定可以预防和治疗 HME 的干预目标。作为必需的细胞内 需氧菌 Ech 的生存依赖于宿主铁。我们发现用预处理人类巨噬细胞 铁螯合剂去铁胺可完全阻断 Ech 感染，表明 Ech 从 宿主不稳定细胞铁（LCI）库。矛盾的是，宿主 LCI 池催化活性氧的形成 物种（ROS），这是宿主防御细胞内病原体的关键机制。我们的项目的 目标是确定 Ech 调节宿主 LCI 池为其获取铁的机制 生长，同时避免 ROS 诱导的宿主细胞反应。通过了解 Ech 获取的过程 铁，我们也许能够预防或限制感染。我们之前发现了 IV 型之间的功能联系 Ech 和宿主细胞的分泌系统（T4SS、VirB/D）、铁和超氧化物歧化物。 铁蛋白含有 重链（FTH）和轻链（FTL）亚基；在我们的初步研究中，我们发现 T4SS 效应器， 埃里希体易位因子(Etf)-3 直接与 FTL 交互，并与 LC3 (ATG8) 共定位，LC3 (ATG8) 是 自噬体。因此，o 我们的中心假设是 Ech 诱导铁蛋白自噬，这是一种选择性的形式 自噬可降解铁蛋白并通过分泌 Etf-3 增加 LCI 池，并安全地捕获铁。我们 将通过三个具体目标来检验我们的中心假设： 目标 1. 分析 Etf-3 和 FTL 之间的相互作用： Etf-3 与人天然铁蛋白的结合动力学； Ech 过程中 Etf-3-FTL 结合的时间模式 感染； Etf-3 与 Ech 感染和未感染细胞中的其他分子结合，包括核受体 共激活剂 4 (NCOA4)；以及 Etf-3 和铁蛋白的细胞共定位。目标 2. 确定 Ech 是否诱导 铁蛋白自噬与通过 T4SS 降低 ROS 同时发生，并且如果需要 Etf-3 诱导的铁蛋白自噬 生产性 Ech 感染；确定 FTL、FTH 和 NCOA4 在 Ech 感染中的作用；比较 Etf-3 诱导的 铁蛋白自噬对 Etf-1 诱导的 Rab5 调节的自噬；确定 NCOA4 是否介导 Etf-3 诱导的 铁蛋白自噬；并绘制诱导铁蛋白自噬的 Etf-3 结构域/片段图。目标 3. 确定是否阻塞 Etf-3 表达和与 FTL 的结合抑制 Ech 诱导的铁蛋白自噬和 Ech 感染。阐明如何 细胞内 Ech 获得铁将 1) 进一步加深我们对细胞内细菌增殖的理解 生存，2）揭示铁稳态的作用，这可能是开发新的关键目标 预防或限制 Ech 感染的方法。如果我们的假设得到支持，结果也将揭示一个独特的 可能被抑制的铁蛋白自噬的分子机制，有利于更广泛的感染领域 疾病和铁稳态。

The gram-negative obligatory intracellular bacterium Ehrlichia chaffeensis (Ech) infects monocytes- macrophages, and causes the emerging tick-borne zoonosis human monocytic ehrlichiosis (HME). Our long- term goal is to identify targets for intervention that can prevent and treat HME. As an obligatory intracellular aerobe, Ech is dependent upon host iron for survival. We found that pretreating human macrophages with the iron chelator deferoxamine resulted in a complete block of Ech infection, suggesting Ech acquires iron from the host labile cellular iron (LCI) pool. Paradoxically, the host LCI pool catalyzes formation of reactive oxygen species (ROS), which is a key mechanism of host defense against intracellular pathogens. Our project's objective is to determine the mechanism by which Ech modulates the host LCI pool to acquire iron for its growth, while averting ROS-induced host cell response. By understanding the process by which Ech acquires iron, we may be able to prevent or limit infection. We previously found functional links between Type IV Secretion System (T4SS, VirB/D), iron, and superoxide dismutatses of Ech and host cells. Ferritin contains heavy chain (FTH) and light chain (FTL) subunits; in our preliminary study we found that the T4SS effector, Ehrlichia translocated factor (Etf)-3 interacts directly with FTL, and colocalizes with LC3 (ATG8), a maker of autophagosomes. Thus, o ur central hypothesis is that Ech induces ferritinophagy, a form of selective autophagy that degrades ferritin and increases the LCI pool by secreting Etf-3, and safely captures iron. We will test our central hypothesis with three Specific Aims: Aim 1. Analyze the interaction between Etf-3 and FTL: Etf-3 binding kinetics to human native ferritin; temporal pattern of Etf-3-FTL binding during the course of Ech infection; Etf-3 binding to other molecules in Ech-infected and uninfected cells, including nuclear receptor coactivator 4 (NCOA4); and cellular co-localization of Etf-3 and ferritin. Aim 2. Determine if Ech induces ferritinophagy that coincides with lowering ROS via T4SS, and if Etf-3-induced ferritinophagy is required for productive Ech infection; determine the roles of FTL, FTH, and NCOA4 in Ech infection; compare Etf-3-induced ferritinophagy to Etf-1-induced Rab5-regulated autophagy; determine if NCOA4 mediates Etf-3-induced ferritinophagy; and map the Etf-3 domains/segments that induce ferritinophagy. Aim 3. Determine if blocking Etf-3 expression and binding to FTL inhibits Ech-induced ferritinophagy and Ech infection. Elucidating how intracellular Ech acquires iron will 1) further our understanding of intracellular bacterial proliferation and survival, and 2) reveal the role of iron homeostasis that may be a critical target for development of new approaches to prevent or limit Ech infection. If our hypothesis is supported, the results will also reveal a unique molecular mechanism of ferritinophagy that may be inhibited, benefiting the broader fields of infectious diseases and iron homeostasis.