Molecular and Cellular Pathogenesis of Legionella

军团菌的分子和细胞发病机制

• 批准号: 9052132
• 负责人: Yousef A Abu Kwaik
• 金额: $ 37.5万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9052132
• 关键词: Alveolar Macrophages; Amoeba genus; Ankyrin Repeat; Asparagine; Bacterial Proteins; Binding; Biogenesis; Bioinformatics; Biological Process; Cell physiology; Cells; Chemicals; Complex; Computer Simulation; Data; Disease; Endoplasmic Reticulum; Environment; Enzymes; Genomics; Genus Mycobacterium; Health; Human; Hydroxylation; Hypoxia; Infection; Inflammatory; Legionella; Legionella pneumophila; Lysosomes; MMP14 gene; Mediating; Membrane; Mixed Function Oxygenases; Modification; Molecular; Morbidity - disease rate; Organism; Pathogenesis; Phagocytes; Pneumonia; Post-Translational Protein Processing; Proliferating; Proteins; Proteome; RNA Interference; Recruitment Activity; Reporting; Role; Shigella; System; Testing; Type IV Secretion System Pathway; Ubiquitination; Vacuole; Yersinia; genome-wide; innovation; killings; knock-down; liquid chromatography mass spectrometry; macrophage; microbial; monocyte; mortality; novel; pathogen; protein protein interaction; transmission process

 DESCRIPTION (provided by applicant): In the aquatic environment, Legionella pneumophila (Lp) proliferates within amoeba but upon transmission to humans it causes pneumonia with high morbidity and mortality. Within amoeba and human macrophages, Lp proliferates within the Legionella-containing vacuole (LCV), which evades lysosomal fusion and is remodeled by the endoplasmic reticulum (ER). Biogenesis of the LCV is controlled by the Dot/Icm type IV secretion system, which injects into the host cell ~300 protein effectors that modulate a myriad of cellular processes to enable intracellular proliferation of Lp. Very few of this large battery o Lp effectors, such as AnkB and AnkH, are required for the intracellular infection. To attain biological function in the host cell, many injected bacterial effectors require post- translational modification by various host machineries. Post-translational modification of eukaryotic proteins by Asparagine (Asn) hydroxylation impacts their protein-protein interaction, and has been widely studied during hypoxic conditions in inflammatory foci. It is not known whether any injected bacterial effector is post-translationally modified by the host asparagine hydroxylase, FIH. The Asn hydroxylation motif has been identified within many human Ankyrin repeats-containing proteins (Ank) that have been shown to be modified by FIH. Our bioinformatics preliminary data have identified an Asn hydroxylation motif within the ankyrin repeats of the AnkB and AnkH effectors of Lp, and we show that both effectors are modified by the host FIH Asparagine hydroxylase. We show that the host FIH is rapidly recruited to the LCV membrane, where AnkH and AnkB are exclusively localized. Our in silico genomic analyses have identified the Asn hydroxylation motif in eight other translocated effectors of Lp. Importantly, RNAi-mediated knockdown or chemical inhibition of FIH abolishes intracellular proliferation of Lp and promotes fusion of the LCV to the lysosomes. Therefore, our hypothesis is: host-mediated Asn hydroxylation of Lp effectors by FIH is required for their functions in biogenesis of the LCV and intracellular proliferation of Lp. To test our hypothesis, our specific aims are to characterize th following: I) Asn hydroxylation of AnkH and its role in the intracellular infection; II) Asn hydroxylation of AnkB and its role in the intracellular infection; and III) Asn hydroxylation of oter Lp effectors and its role in biogenesis of the LCV. Upon completion of our proposed studies, the biological function and host targets of AnkH will be known, and the ubiquitination substrates of AnkB will be identified. The role of host FIH-mediated Asn hydroxylation in the biological function of AnkH and AnkB will be uncovered. This is significant, since both effectors are two of very few of the battery of ~300 Lp effectors to be required for intra-vacuolar proliferation. We wil report on our novel discovery of the acquisition of the FIH complex by a pathogen-containing vacuole and its role in modification of several Lp effectors. We will uncover the role of FIH-mediated Asn hydroxylation of several Lp effectors in lysosomal evasion by the LCV. Our proposed studies on Lp-primary hMDMs interaction and specific manipulation of the host post- translational modification by FIH are innovative with broad high impact and significance not only for Lp but for other pathogens. This is evident from our discovery of Asn hydroxylation of the YopM effector of Yersinia, and potential Asn hydroxylation of the IpaH4.5 effector of Shigella, and the EsxF effector of Mycobacterium.

 描述（由申请方提供）：在水生环境中，嗜肺军团菌（Lp）在变形虫体内增殖，但在传播给人类后会引起肺炎，发病率和死亡率较高。在阿米巴和人巨噬细胞内，Lp在含有军团菌的空泡（LCV）内增殖，其逃避溶酶体融合并被内质网（ER）重塑。LCV的生物发生由Dot/Icm IV型分泌系统控制，该系统将调节无数细胞过程的~300种蛋白质效应物注入宿主细胞中以使Lp能够在细胞内增殖。这种大的Lp效应子电池中的很少一部分，如AnkB和AnkH，是细胞内感染所需的。为了在宿主细胞中获得生物学功能，许多注射的细菌效应子需要翻译后修饰。 各种主机的修改。天冬酰胺（Asn）羟基化对真核蛋白质的翻译后修饰影响其蛋白质-蛋白质相互作用，并已在炎症灶的缺氧条件下被广泛研究。目前尚不清楚任何注射的细菌效应物是否被宿主天冬酰胺羟化酶FIH进行了后修饰。Asn羟基化基序已在许多人锚蛋白重复序列蛋白（Ank）中被鉴定，这些蛋白已被FIH修饰。我们的生物信息学的初步数据已经确定了一个天冬酰胺羟基化基序内的锚蛋白重复的AnkB和AnkH的Lp效应，我们表明，这两个效应器被修改的主机FIH天冬酰胺羟化酶。我们表明，主机FIH迅速招募到LCV膜，其中AnkH和AnkB是专门本地化。我们的计算机基因组分析已经确定了天冬酰胺羟基化基序在其他八个易位的Lp效应。重要的是，FIH的RNAi介导的敲低或化学抑制消除了Lp的细胞内增殖并促进LCV与溶酶体的融合。因此，我们的假设是：通过FIH的Lp效应物的宿主介导的Asn羟基化是它们在LCV的生物发生和Lp的细胞内增殖中的功能所必需的。为了验证我们的假设，我们的具体目标是表征以下：I）AnkH的Asn羟基化及其在细胞内感染中的作用; II）AnkB的Asn羟基化及其在细胞内感染中的作用;以及III）其它Lp效应物的Asn羟基化及其在LCV的生物发生中的作用。在我们的研究完成后，AnkH的生物学功能和宿主靶点将是已知的，AnkB的泛素化底物将被确定。将揭示宿主FH介导的Asn羟基化在AnkH和AnkB生物学功能中的作用。这是重要的，因为这两种效应子是液泡内增殖所需的约300种Lp效应子中的两种。我们将报告我们的新发现的收购FIH复合物的病原体含有空泡和它的作用，修改几个脂蛋白效应。我们将揭示的作用FIH介导的天冬酰胺羟基化的几个Lp效应器在溶酶体逃避LCV。我们提出的关于脂蛋白与原发性hMDM相互作用以及FIH对宿主翻译后修饰的特异性操纵的研究是创新的，不仅对脂蛋白而且对其他病原体都具有广泛的高度影响和意义。这从我们发现耶尔森氏菌的YopM效应子的Asn羟基化、志贺氏菌的IpaH4.5效应子的潜在Asn羟基化和分枝杆菌的EsxF效应子中是明显的。