Structure and Function of Legionella pneumophila Lysine Methyltransferases

嗜肺军团菌赖氨酸甲基转移酶的结构和功能

• 批准号: 10445910
• 负责人: RAYMOND C TRIEVEL
• 金额: $ 53.11万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-21 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10445910
• 关键词: Address; Affect; Agreement; American; Amino Acids; Amoeba genus; Ankyrin Repeat; Architecture; Bacterial Pneumonia; Binding; Biochemical; Biochemistry; Biocide; Biological; Biological Assay; Biological Process; C-terminal; Cell Death; Cell Nucleus; Cells; Chromatin; Chronic lung disease; Complement; Complex; Coupled; Cryoelectron Microscopy; Crystallization; DNA; Data; Defect; Disease; Distal; Elderly; Elements; Enzymes; Etiology; Exhibits; Fatality rate; Foundations; Gene Deletion; Gene Expression; Gene Expression Regulation; Genes; Histone H3; Histone-Lysine N-Methyltransferase; Histones; Image; Immune; Immune response; Immunocompromised Host; Impairment; In Vitro; Individual; Infection; Innate Immune Response; Kinetics; Laboratories; Legionella; Legionella pneumophila; Legionnaires' Disease; Lung; Lysine; Mediating; Membrane; Methylation; Methyltransferase; Michigan; Microbiology; Modification; Molecular; Morbidity - disease rate; Mutagenesis; Mutation; N-terminal; Natural Immunity; Nuclear; Nucleosomes; Orthologous Gene; Paris, France; Pathogenesis; Pathogenicity; Peptides; Proteins; Reader; Roentgen Rays; Role; S-Adenosylmethionine; SET Domain; Signal Pathway; Signal Transduction; Site; Specificity; Structure; Substrate Specificity; Tail; Testing; Universities; Virulence; atypical pneumonia; base; chromatin modification; combat; gene repression; genome-wide; inhibitor; insight; interdisciplinary collaboration; macrophage; molecular pathology; mortality; mutant; nanomolar; novel; novel therapeutics; particle; pathogen; pathogenic bacteria; structural biology; therapeutic development; trafficking

PROJECT ABSTRACT Legionella pneumophila is a intracellular bacterial pathogen that is the primary etiological agent of Legionnaires’ Disease. This disease is categorized as an atypical pneumonia that afflicts the elderly and individuals who are immunocompromised or suffer from chronic lung disease. L. pneumophila infects host cells, such as environmental amoeba and lung macrophages, through the secretion of an array of effector proteins that subvert signaling pathways, membrane trafficking, and gene expression in host cells to promote bacterial replication. Among these effectors are the protein lysine methyltransferases (KMTs) RomA and its highly conserved strain ortholog LegAS4. RomA has been shown to localize to the nucleus where it trimethylates Lys14 in histone H3 (H3K14me3). This novel chromatin modification silences the macrophage gene expression, including loci responsible for innate immunity. In agreement with these findings, deletion of the RomA gene in L. pneumophila hinders bacterial replication within infected host cells, illustrating the importance of this KMT in pathogenesis. Despite the role of RomA in L. pneumophila virulence, outstanding questions regarding the biological functions of RomA and LegAS4 remain unresolved. These questions include the molecular basis of their unique substrate specificity for H3K14, how they interact with chromatin, and the biological roles of LegAS4 in host cell infection. The Trievel, Swanson, Cho, and Ohi laboratories at the University of Michigan have established an interdisciplinary collaboration to address these questions by employing an integrated approach combining biochemistry, microbiology, and structural biology. Our overall objective is to elucidate the mechanism by which RomA and LegAS4 recognize and methylate H3K14 in nucleosomes in vitro and in host cells infected by L. pneumophila. We propose the following specific aims to accomplish this objective: 1) Define the molecular mechanism of H3K14 methylation by RomA and LegAS4. 2) Elucidate the structural basis of nucleosome recognition. 3) Determine the functions of LegAS4 in macrophage infection. We envision that these studies will yield a comprehensive framework for understanding the mechanisms underlying nucleosome recognition and H3K14 methylation by RomA and LegAS4 that promote L. pneumophila replication during host cell infection. These results will provide avenues for developing selective inhibitors of these KMTs that can be applied as novel therapeutics or biocides to combat Legionnaires Disease.

项目摘要 嗜肺军团菌是一种细胞内细菌病原体，是军团菌性肺炎的主要病原体。 疾病这种疾病被归类为非典型肺炎，影响老年人和 免疫力低下或患有慢性肺病。L.嗜肺菌感染宿主细胞，例如 环境阿米巴和肺巨噬细胞，通过分泌一系列的效应蛋白，破坏 信号传导途径、膜运输和宿主细胞中的基因表达以促进细菌复制。 在这些效应子中有蛋白质赖氨酸甲基转移酶（KMTs）RomA及其高度保守的菌株 直系同源物LegAS 4。RomA已被证明定位于细胞核，在那里它使组蛋白H3中的Lys14三甲基化 （H3K14me3）。这种新的染色质修饰沉默巨噬细胞基因表达，包括基因座 负责先天免疫。与这些发现一致，在L.嗜肺 阻碍细菌在感染的宿主细胞内复制，说明这种KMT在发病机制中的重要性。 尽管罗马人在L.嗜肺菌毒力，关于生物学功能的未决问题 RomA和LegAS4的问题仍未解决。这些问题包括它们独特底物的分子基础 H3K14的特异性，它们如何与染色质相互作用，以及LegAS4在宿主细胞感染中的生物学作用。 密歇根大学的Trievel，Swanson，Cho和Ohi实验室已经建立了一个 跨学科合作通过采用综合方法来解决这些问题，该方法结合 生物化学、微生物学和结构生物学。我们的总体目标是阐明 RomA和LegAS4识别并甲基化体外核小体中的H3K14和在被L. 嗜肺菌我们建议实现这一目标的具体目标如下： 1)通过RomA和LegAS4定义H3K14甲基化的分子机制。 2)阐明核小体识别的结构基础。 3)确定LegAS4在巨噬细胞感染中的功能。 我们设想，这些研究将产生一个全面的框架，了解机制， 潜在的核小体识别和H3K14甲基化的RomA和LegAS4，促进L.嗜肺 在宿主细胞感染期间复制。这些结果将为开发选择性抑制剂提供途径。 这些KMT可以作为新的治疗剂或杀生物剂用于对抗军团病。