Structure-function analysis of a catalase/ankyrin tandem from Pseudomonas aeruginosa necessary for resistance to hydrogen peroxide

铜绿假单胞菌过氧化氢酶/锚蛋白串联的结构-功能分析对于抵抗过氧化氢是必需的

• 批准号: 10598215
• 负责人: Rhett Kovall
• 金额: $ 24.3万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-11-08 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10598215
• 关键词: Ankyrin Repeat; Ankyrins; Antibiotic Resistance; Behavior; Binding; Biochemical; Biological Assay; Biological Process; Biophysics; Burn injury; Chronic Obstructive Pulmonary Disease; Clinical; Cysteine; Cystic Fibrosis; Cytoplasm; DNA Binding; DNA Damage; Data; Disease; Drug Metabolic Detoxication; Escherichia coli; Eukaryota; Exposure to; Fractionation; Genes; Genetic; Genetic Transcription; Goals; Gram-Negative Bacteria; Heme; Hydrogen Peroxide; Immune response; In Vitro; Infection; Innate Immune Response; Innate Immune System; Link; Macrophage; Mediating; Medical; Microbial Biofilms; Microbial Physiology; Modeling; Multi-Drug Resistance; Nosocomial Infections; Operative Surgical Procedures; Operon; Oxidants; Periplasmic Proteins; Phagocytes; Prokaryotic Cells; Proteins; Proton-Motive Force; Pseudomonas aeruginosa; Pseudomonas aeruginosa infection; Recombinants; Resistance; Respiratory Burst; Roentgen Rays; Role; Structure; Testing; Trans-Activators; Urinary tract infection; bactericide; catalase; design; diabetic ulcer; disulfide bond; heme 1; heme a; heme-binding protein; human disease; human pathogen; human tissue; in vitro testing; in vivo; insight; interdisciplinary approach; monomer; mutant; neutrophil; novel; opportunistic pathogen; pathogenic bacteria; periplasm; protein protein interaction; protein purification; public health relevance; response; translational study

PROJECT SUMMARY/ABSTRACT Pseudomonas aeruginosa (PA) is a ubiquitous Gram-negative bacteria and opportunistic pathogen that is of considerable medical importance due to its multidrug resistance and tendency to form antibiotic resistant biofilms. During PA infection, a strong innate immune response is generated by phagocytes, including neutrophils and macrophages, in the form of the respiratory burst (RB). Although many bactericidal agents are generated during the RB, a leading DNA-damaging and PMF (proton motive force) perturbing oxidant is hydrogen peroxide (H2O2). PA is exposed to phagocyte-derived H2O2 in a number of disease settings, including cystic fibrosis, chronic obstructive pulmonary disease, burns/blast/surgical/diabetic wounds and urinary tract infections. PA defense against H2O2 is governed by the DNA-binding transactivator OxyR. Of the numerous PA genes under OxyR control, optimal resistance to H2O2 requires activation of a two gene operon encoding katb and ankb, which encode two proteins strategically deployed to the periplasm. KatB is a group 1 heme b-type catalase that converts H2O2 into H2O and O2 and AnkB is a putative ankyrin repeat protein of unknown function. The overall goal of this multi-PI exploratory R21 application is to combine the structural, biophysical, and biochemical expertise of the Kovall lab with the microbial physiology, biofilm, and genetics expertise of the Hassett lab in order to elucidate the novel functions of AnkB and KatB in response to exogenous H2O2. Our preliminary data supports the hypothesis that AnkB is a novel heme binding protein that is required to transfer heme to monomeric KatB in order to facilitate the formation of catalytically active KatB tetramers. This mechanism is likely an important feature of the in vivo confrontation between PA and the H2O2 component of the RB mediated by phagocytic cells during PA infection. To achieve our overall goal and test our hypothesis, we will pursue the following two specific aims. Aim 1: Determine the structures and define the functions of AnkB and KatB. Aim2: Determine the role of AnkB and KatB in planktonic vs. biofilm-forming PA in response to H2O2. Completion of these exploratory, structural, mechanistic, and highly translational studies will begin to uncover the role of AnkB and KatB in protecting PA from H2O2 and will likely lead to important functional insights into the overall behavior of PA during infection associated with various important human diseases.

项目总结/摘要 铜绿假单胞菌（PA）是一种普遍存在的革兰氏阴性菌和条件致病菌， 由于其多药耐药性和形成抗生素耐药性的倾向， 生物膜在PA感染期间，吞噬细胞产生强烈的先天免疫应答，包括 中性粒细胞和巨噬细胞，以呼吸爆发（RB）的形式。尽管许多杀菌剂 在RB过程中产生，一种主要的DNA损伤和PMF（质子动力）干扰氧化剂， 过氧化氢（H2 O2）。PA在许多疾病环境中暴露于吞噬细胞衍生的H2 O2，包括 囊性纤维化、慢性阻塞性肺病、烧伤/爆炸/手术/糖尿病伤口和泌尿道 感染. PA对H2 O2的防御由DNA结合反式激活剂OxyR控制。在众多的PA 在OxyR控制下，对H2 O2的最佳抗性需要激活编码katb的两个基因操纵子 和ankb，其编码两种策略性地部署到周质的蛋白质。KatB是一种1组血红素b型 过氧化氢酶将H2 O2转化为H2O和O2，AnkB是一种功能未知的推定锚蛋白重复蛋白。 这种多PI探索性R21应用的总体目标是将结构、生物物理和生物物理特性联合收割机结合起来。 科瓦尔实验室的生物化学专业知识与微生物生理学，生物膜，和遗传学专业知识， 为了阐明AnkB和KatB在响应外源H2 O2时的新功能，Hassett实验室进行了研究。我们 初步数据支持AnkB是一种新的血红素结合蛋白的假设， 将血红素转化为单体KatB以促进催化活性KatB四聚体的形成。这 机制可能是PA和H2 O2组分之间体内对抗的重要特征。 在PA感染期间由吞噬细胞介导的RB。为了实现我们的总体目标并验证我们的假设，我们 将致力于实现以下两个具体目标。目的1：确定AnkB的结构和功能， KatB.目的2：确定AnkB和KatB在对H2 O2的反应中的促渗透作用与生物膜形成作用。 完成这些探索性的、结构性的、机械的和高度转化的研究将开始揭示 AnkB和KatB在保护PA免受H2 O2侵害中的作用，并可能导致对PA的重要功能性见解。 PA在与各种重要人类疾病相关的感染期间的总体行为。