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

项目摘要/摘要 铜绿假单胞菌(PA)是一种普遍存在的革兰氏阴性杆菌和条件致病菌。 由于其多重耐药性和形成抗生素耐药性的趋势，具有相当大的医学价值 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。尽管许多杀菌剂是 在RB过程中产生的一种主要的DNA损伤和PMF(质子动力)扰动氧化剂是 过氧化氢(过氧化氢)。PA在许多疾病环境中暴露于吞噬细胞来源的过氧化氢，包括 囊性纤维化、慢性阻塞性肺疾病、烧伤/原始/外科/糖尿病伤口和尿路 感染。PA对H_2O_2的防御是由DNA结合的反式激活因子OxyR控制的。在众多的PA中 在OxyR控制下的基因，对过氧化氢的最佳抗性需要编码katb的两个基因操纵子的激活 和ankb，它编码两种蛋白质，战略性地部署到周质中。Katb是第一组血红素b型 过氧化氢酶将H_2O_2和O_2转化为H_2O和O_2，AnkB是一种功能未知的锚蛋白重复蛋白。 这种多PI探索性R21应用程序的总体目标是将结构、生物物理和 Kovall实验室的生物化学专业知识和微生物生理学、生物膜和遗传学专业知识 为了阐明AnkB和KatB在响应外源H_2O_2时的新功能。我们的 初步数据支持AnkB是一种新的血红素结合蛋白的假设，需要转移 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与H_2O_2组分体内对抗的一个重要特征 PA感染过程中吞噬细胞介导的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的结构和功能 卡特布。目的：确定AnkB和KatB在浮游生物与生物膜形成PA对H_2O_2反应中的作用。 这些探索性的、结构性的、机械性的和高度翻译的研究的完成将开始揭示 AnkB和KatB在保护PA免受H_2O_2侵袭中的作用，可能会导致对 PA在感染期间的总体行为与各种重要的人类疾病有关。

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.