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

• 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
• 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

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.

项目摘要 莱姆病的病原体伯氏疏螺旋体，很好地适应其多种蜱， 哺乳动物宿主，尽管最低限度的基因组使其缺乏许多功能，但它已经高度进化， 有效的机制，以逃避不同的主机防御。作为在这些不同宿主中生存的一部分， 通常的要求是中和氧化和亚硝化应激反应的能力。B。布格多费里湖 活性氧（ROS）和活性氮（RNS）解毒酶的显著缺乏 在大多数细菌病原体中发现，这表明它使用新的机制来逃避这些防御。在我们 根据初步数据，我们已经鉴定了30多个新基因，它们似乎与B有关。伯氏抗性 ROS或RNS。通过将转座子文库暴露于ROS或RNS胁迫来鉴定基因 随后测序（Tn-seq）以精确确定单个突变体的适合度。被识别基因 使用单个突变体和互补突变体证实。被鉴定为具有 在ROS和/或RNS抗性中的作用，测试它们对它们的天然蜱和小鼠宿主的感染性。我们 鉴定了对小鼠或蜱具有感染性缺陷的多种突变体。 在这个提议中，我们试图理解B使用的独特机制。伯氏菌以避免氧化 和亚硝化杀人我们提出以下三个具体目标。首先，在目标1中，我们将评估 B的ROS/RNS反应蛋白。在哺乳动物感染过程中的生存。同时使用 突变的细菌和敲除小鼠缺乏ROS/RNS防御的特定成分，我们将解剖 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， 和定量RT-PCR来确定单个基因的作用。接下来，在目标2中，我们将评估 B的ROS/RNS反应蛋白。burgdorferi在其蜱宿主中的生存使用与Aim 1相似的策略。 最后，在目标3中，我们将确定候选蛋白质如何介导对ROS的抗性。在这个目标中，我们将 测试几种靶蛋白在ROS/RNS解毒中的作用的预测活性。 这项研究代表了第一个全球评估B。burgdorferi对抗ROS/RNS。 考虑到这种应答在宿主先天免疫中的重要性，这些疏螺旋体的特征在于它们的抗原性。 基因/蛋白质将提供重要的洞察能力的B。伯氏为了避免清除和坚持 在自然界中它们占据的宿主体内长时间存在。深入了解生物体 能够逃脱免疫介导的杀伤可能对理解人类的免疫系统具有重要意义。 莱姆病