Keys to prevent iron hijacking and infection by intracellular bacteria

防止铁劫持和细胞内细菌感染的关键

• 批准号: 10330564
• 负责人: YASUKO RIKIHISA
• 金额: $ 39万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10330564
• 关键词: Aerobic Bacteria; Amino Acids; Animal Diseases; Autophagocytosis; Autophagosome; Back; Bacteria; Binding; Cells; Communicable Diseases; Complementary DNA; Cytoplasm; Deferoxamine; Development; Doxycycline; Ehrlichia; Ehrlichia chaffeensis; Ehrlichiosis; Emerging Communicable Diseases; Ferritin; Future; Goals; Growth; Homeostasis; Host Defense Mechanism; Human; Hybrids; Immunologics; Infection; Intervention; Iron; Iron Chelating Agents; Iron Superoxide Dismutase; Kinetics; Knock-in; Knowledge; Leukocytes; Libraries; Light; Link; Maps; Mediating; Membrane; Metabolism; Mission; Mitochondria; Molecular; Nuclear Receptor Coactivator 4; Organelles; Oxides; Pathogenesis; Pattern; Peptide Nucleic Acids; Pharmacology; Prevalence; Preventive measure; Process; Proteins; Public Health; Reactive Oxygen Species; Rickettsia; Risk; Role; SOD2 gene; Signal Pathway; Small Interfering RNA; Superoxides; Surface; Surface Plasmon Resonance; Testing; Therapeutic; Ticks; Time; Tissues; Transfection; Type IV Secretion System Pathway; United States National Institutes of Health; Work; Yeasts; base; cell injury; disability; human disease; innovation; iron metabolism; knock-down; macrophage; monocyte; mutant; nanobodies; novel; novel strategies; pathogen; prevent; response; screening; tick-borne

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.

革兰氏阴性的细胞内细菌Chaffeensis（ECH）感染单核细胞 巨噬细胞，并引起新兴的tick传播人畜共患病人类单核细胞增多症（HME）。我们的长期 术语目标是确定可以预防和治疗HME的干预目标。作为强制性细胞内 Aerobe，ECH取决于宿主铁的生存。我们发现，通过 铁螯合剂脱氧胺导致ECH感染的完整块，表明ECH从 宿主不稳定的细胞铁（LCI）池。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 目的是确定ECH调节主机LCI池以获取铁的机制 生长，同时避免ROS诱导的宿主细胞反应。通过了解ECH获得的过程 铁，我们可能能够预防或限制感染。我们先前发现了IV型之间的功能链接 分泌系统（T4SS，VIRB/D），ECH和宿主细胞的铁和超氧化物歧义。 铁蛋白包含 重链（FTH）和轻链（FTL）亚基；在我们的初步研究中，我们发现T4SS效应子， Ehrlichia易位因子（ETF）-3 直接与FTL相互作用，并与LC3（ATG8）共定位， 自噬体。因此，o UR中心假设是ECH诱导了铁蛋白噬菌体，这是一种选择性的形式 自噬通过分泌ETF-3来降解铁蛋白并增加LCI池并安全捕获铁。我们 将以三个特定目的测试我们的中心假设：目标1。分析ETF-3和FTL之间的相互作用： ETF-3结合动力学与人类生产铁蛋白； ECH过程中ETF-3-FTL结合的时间模式 感染; ETF-3与ECH感染和未感染的细胞中的其他分子结合，包括核受体 共激活因子4（NCOA4）; ETF-3和铁蛋白的细胞共定位。 AIM 2。确定ECH是否引起 铁毒素与通过T4SS降低ROS相吻合，如果需要ETF-3诱导的铁蛋白嗜酸剂 生产性的感染；确定FTL，FTH和NCOA4在ECH感染中的作用；比较ETF-3诱导 铁蛋白噬菌至ETF-1诱导的RAB5调节的自噬；确定NCOA4是否介导ETF-3诱导的 铁毒素;并绘制诱导铁毒素的ETF-3域/段。目标3。确定是否阻止 ETF-3表达并与FTL结合抑制了ECH诱导的铁毒素和ECH感染。阐明如何 细胞内ECH获得铁将1）进一步了解细胞内细菌增殖和 生存和2）揭示铁稳态的作用，这可能是发展新的关键目标 预防或限制ECH感染的方法。如果支持我们的假设，结果也将揭示一个独特的 铁毒素的分子机制可能被抑制，从而使更广泛的传染性领域受益 疾病和铁稳态。