Keys to prevent iron hijacking and infection by intracellular bacteria

革兰氏阴性专性细胞内细菌查菲埃里希体（Ech）感染单核细胞- 巨噬细胞，并导致新出现的蜱传人畜共患病人单核细胞埃里希体病（HME）。我们长久以来- 长期目标是确定可以预防和治疗HME的干预目标。作为一种强制性的细胞内 Ech依赖于宿主铁来生存。我们发现， 铁螯合剂去铁胺导致Ech感染的完全阻断，表明Ech从 宿主不稳定细胞铁库。特别地，宿主LCI池催化活性氧的形成， 活性氧（ROS）是宿主防御胞内病原体的关键机制。我们的项目 目的是确定Ech调节宿主LCI池以获得铁用于其代谢的机制。 生长，同时避免ROS诱导的宿主细胞反应。通过了解Ech获得 铁，我们也许可以预防或限制感染。我们以前发现IV型糖尿病与 分泌系统（T4 SS，VirB/D），铁，超氧化物歧化酶的Ech和宿主细胞。 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T4 SS效应子， 埃里希体易位因子-3 直接与FTL相互作用，并与LC 3（ATG 8）共定位，LC 3（ATG 8）是一种 自噬体因此，o 我们的中心假设是，Ech诱导铁蛋白吞噬，一种选择性的 自噬，其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 我们将用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与感染和未感染细胞中的其他分子结合，包括核受体 共激活因子4（NCOA 4）;和Etf-3和铁蛋白的细胞共定位。目标二。确定Ech是否诱导 铁蛋白吞噬与通过T4 SS降低ROS相一致，并且如果Etf-3诱导的铁蛋白吞噬是 生产性Ech感染;确定FTL，FTH和NCOA 4在Ech感染中的作用;比较Etf-3诱导的Ech感染。 铁蛋白吞噬对Etf-1诱导的Rab 5调节的自噬;确定NCOA 4是否介导Etf-3诱导的自噬。 并绘制诱导铁蛋白吞噬的Etf-3结构域/区段。目标3：确定是否阻塞 Etf-3表达和结合FTL抑制Ech-induced ferritinophagy和Ech-infection。阐明如何 细胞内Ech获得铁将1）进一步我们对细胞内细菌增殖的理解， 生存，和2）揭示铁稳态的作用，这可能是一个新的发展的关键目标， 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吞噬的分子机制，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.