Coping with Stress: Next Generation Approaches to Borrelia burgdorferi Host Adaptation

应对压力：伯氏疏螺旋体宿主适应的下一代方法

基本信息

批准号：
9892949
负责人：
Linden T Hu
金额：
$ 66.15万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-04-01 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9892949
关键词：
Animal Model Animals Antioxidants Arthropods Bacteria Birds Bite Blood Borrelia Borrelia burgdorferi Borrelia oxidative stress regulator Cells Collaborations Collection Complement Coupling Data Defect Deletion Mutagenesis Drug Metabolic Detoxication Elements Enzymes Equilibrium Escherichia coli Etiology Event Exhibits Exposure to Ferritin Gene Proteins Genes Genetic Screening Genome Goals Host Defense Human Image Immune In Vitro Individual Infection Invaded Knockout Mice Libraries Link Lyme Disease Mammals Maps Mediating Metabolic Pathway Metals Molting Mus Natural Immunity Nature Nymph Organism Oxidation-Reduction Oxidative Stress Oxidoreductase Pathogenesis Pathogenicity Peroxidases Phase Phenotype Physiological Proteins Reactive Nitrogen Species Reactive Oxygen Species Recombinant Proteins Resistance Role Site Stress Study models Superoxides Technology Testing Ticks Time Vasodilation bacterial fitness biological adaptation to stress catalase cell injury cellular targeting combat coping experimental study fitness genetic technology in vitro Assay in vivo in vivo imaging insight mutant next generation next generation sequencing nitrogen compounds nitrosative stress novel oxygen compounds pathogen pathogenic bacteria predictive test response soft drink success transcriptome sequencing transmission process transposon sequencing

项目摘要

PROJECT SUMMARY Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, is well adapted to its multiple tick and mammalian hosts and, despite a minimalist genome leaving it devoid of many functions, has evolved highly effective mechanisms for evading different host defenses. As a part of survival within such disparate hosts, one common requirement is the ability to neutralize oxidative and nitrosative stress responses. B. burgdorferi have a striking dearth of reactive oxygen species (ROS) and reactive nitrogen species (RNS) detoxification enzymes seen in most bacterial pathogens suggesting that it uses novel mechanisms to evade these defenses. In our preliminary data, we have identified over 30 new genes that appear to be involved in B. burgdorferi resistance to ROS or RNS. Genes were identified by exposure of a transposon library to either ROS or RNS stress followed by sequencing (Tn-seq) to precisely determine the fitness of individual mutants. Identified genes were confirmed using individual mutants and complemented mutants. Mutants in genes identified as having a role in ROS and/or RNS resistance were tested for their infectivity into their natural tick and mouse hosts. We identified multiple mutants with defects in infectivity into mice or ticks. In this proposal, we seek to understand the unique mechanisms used by B. burgdorferi to evade oxidative and nitrosative killing. We are proposing the following 3 Specific Aims. First, in Aim 1, we will assess the role of ROS/RNS responsive proteins of B. burgdorferi for survival during mammalian infection. Using both mutant bacteria and knockout mice devoid of specific components of the ROS/RNS defenses, we will dissect the role of specific genes at different time points in the infectious cycle. We will use in vivo imaging, RNA-seq and quantitative rt-PCR to pinpoint the role of individual genes. Next, in Aim 2, we will assess the role of ROS/RNS responsive proteins of B. burgdorferi for survival in its tick host using similar strategies to Aim 1. Finally, in Aim 3, we will determine how the candidate proteins mediate resistance to ROS. In this Aim we will test the predicted activity of several targeted proteins for their role in detoxification of ROS/RNS. This study represents the first genetic screen to globally assess how B. burgdorferi combats ROS/RNS. Given the importance of this response in host innate immunity, the characterization of these borrelial genes/proteins will provide important insight into the ability of B. burgdorferi to avoid clearance and persist for prolonged periods within the hosts they occupy in nature. Insights into mechanisms by which the organism is able to escape immune mediated killing are likely to have important implications for understanding human Lyme disease.

项目概要伯氏疏螺旋体（Borrelia burgdorferi）是莱姆疏螺旋体病的病原体，非常适应其多种蜱虫的存在哺乳动物宿主，尽管基因组极简主义使其缺乏许多功能，但已经高度进化逃避不同宿主防御的有效机制。作为在如此不同的宿主中生存的一部分，一个共同的要求是中和氧化和亚硝化应激反应的能力。伯氏疏螺旋体有活性氧 (ROS) 和活性氮 (RNS) 解毒酶的严重缺乏在大多数细菌病原体中都可以看到，这表明它使用新的机制来逃避这些防御。在我们的初步数据显示，我们已经鉴定出 30 多个新基因，这些基因似乎与伯氏疏螺旋体抗性有关到 ROS 或 RNS。通过将转座子文库暴露于 ROS 或 RNS 胁迫来鉴定基因然后进行测序 (Tn-seq) 以精确确定单个突变体的适合度。已鉴定基因使用个体突变体和互补突变体证实了这一点。基因突变被鉴定为具有测试了它们在 ROS 和/或 RNS 抗性中的作用，以确定它们对天然蜱和小鼠宿主的感染性。我们鉴定出多个对小鼠或蜱具有感染性缺陷的突变体。在本提案中，我们试图了解伯氏疏螺旋体用来逃避氧化的独特机制和亚硝化杀灭。我们提出以下 3 个具体目标。首先，在目标 1 中，我们将评估角色伯氏疏螺旋体的 ROS/RNS 响应蛋白在哺乳动物感染期间的存活。两者同时使用突变细菌和基因敲除小鼠缺乏 ROS/RNS 防御的特定成分，我们将剖析特定基因在感染周期不同时间点的作用。我们将使用体内成像、RNA-seq 以及定量 rt-PCR 来查明单个基因的作用。接下来，在目标 2 中，我们将评估使用与目标 1 类似的策略，伯氏疏螺旋体的 ROS/RNS 响应蛋白在其蜱宿主中存活。最后，在目标 3 中，我们将确定候选蛋白如何介导 ROS 抗性。为了这个目标，我们将测试几种目标蛋白在 ROS/RNS 解毒中的作用的预测活性。这项研究代表了首次在全球范围内评估伯氏疏螺旋体如何对抗 ROS/RNS 的基因筛查。鉴于这种反应在宿主先天免疫中的重要性，这些疏螺旋体的特征基因/蛋白质将为了解伯氏疏螺旋体避免清除和持续存在的能力提供重要的见解长期生活在它们在自然界中占据的宿主体内。深入了解有机体的机制能够逃脱免疫介导的杀伤可能对理解人类具有重要意义莱姆病。