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.

项目概要/摘要 铜绿假单胞菌 (PA) 是一种普遍存在的革兰氏阴性细菌和机会性病原体， 由于其多重耐药性和形成抗生素耐药性的倾向，具有相当大的医学重要性 生物膜。在 PA 感染期间，吞噬细胞会产生强烈的先天免疫反应，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 过程中产生的一种主要的 DNA 损伤性和 PMF（质子动力）干扰氧化剂是 过氧化氢（H2O2）。在许多疾病中，PA 都会暴露于吞噬细胞衍生的 H2O2，包括 囊性纤维化、慢性阻塞性肺病、烧伤/爆炸/手术/糖尿病伤口和尿路 感染。 PA 对 H2O2 的防御是由 DNA 结合反式激活因子 OxyR 控制的。在众多的 PA 中 OxyR 控制下的基因，对 H2O2 的最佳抗性需要激活编码 katb 的双基因操纵子 和 ankb，编码两种策略性部署到周质的蛋白质。 KatB 是 1 族血红素 B 型 过氧化氢酶将 H2O2 转化为 H2O 和 O2，AnkB 是一种假定的功能未知的锚蛋白重复蛋白。 这个多 PI 探索性 R21 应用的总体目标是将结构、生物物理和 Kovall 实验室的生化专业知识与微生物生理学、生物膜和遗传学专业知识 Hassett 实验室旨在阐明 AnkB 和 KatB 响应外源 H2O2 的新功能。我们的 初步数据支持这样的假设：AnkB 是一种新型血红素结合蛋白，需要进行转移 将血红素转化为单体 KatB，以促进具有催化活性的 KatB 四聚体的形成。这 机制可能是 PA 与 H2O2 成分在体内对抗的一个重要特征。 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检验我们的假设，我们 将追求以下两个具体目标。目标1：确定AnkB和AnkB的结构并定义其功能 凯特B。目标 2：确定 AnkB 和 KatB 在浮游 PA 与生物膜形成 PA 响应 H2O2 中的作用。 完成这些探索性、结构性、机制性和高度转化性的研究将开始揭示 AnkB 和 KatB 在保护 PA 免受 H2O2 侵害方面的作用，可能会导致对 PA 在与各种重要人类疾病相关的感染期间的总体行为。