Keys to prevent iron hijacking and infection by intracellular bacteria

革兰氏阴性强制胞内细菌查菲埃利希菌(Ech)感染单核细胞- 巨噬细胞，并引起人单核细胞埃立克体病(HME)。我们的长- 长期目标是确定可以预防和治疗HME的干预目标。作为一个强制性的细胞内 需氧菌，Ech依赖宿主铁生存。我们发现，用人巨噬细胞预处理 铁螯合剂去铁胺导致Ech感染完全阻断，表明Ech从 宿主不稳定细胞铁(LCI)池。矛盾的是，宿主LCI池催化形成活性氧 ROS是寄主抵御细胞内病原菌的关键机制。我们的项目 目的是确定Ech调节宿主LCI池为其提供铁的机制 生长，同时避免ROS诱导的宿主细胞反应。通过了解Ech收购的过程 铁，我们或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。 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， 埃立克体易位因子-3 直接与FTL交互，并与以下制造商LC3(ATG8)协同定位 自噬小体。因此，o 我们的中心假设是，Ech诱导了吞铁素，这是选择性的一种形式 自噬，通过分泌ETF-3降解铁蛋白并增加LCI池，并安全地捕获铁。我们 我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Ech感染和未感染细胞中的其他分子结合，包括核受体 辅活化子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 与通过T4SS降低ROS一致的铁质吞噬，以及如果需要ETF-3诱导的铁质吞噬 产生性Ech感染；确定FTL、FTH和NCOA4在Ech感染中的作用；比较ETF-3诱导的 铁蛋白对ETF-1诱导的Rab5调节的自噬作用；确定NCOA4是否介导了ETF-3诱导的自噬 并定位了诱导铁素噬菌体的ETF-3结构域/片段。目标3.确定阻止 ETF-3的表达和与FTL的结合可抑制Ech诱导的铁蛋白吞噬和Ech感染。阐明如何 细胞内的Ech获得铁将1)进一步了解细胞内细菌的增殖和 生存，以及2)揭示铁稳态的作用，这可能是新的开发的关键靶点 预防或限制Ech感染的方法。如果我们的假设得到支持，结果也将揭示出一种独特的 噬铁蛋白的分子机制可能被抑制，有益于更广泛的感染性领域 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.