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.

项目总结 伯氏疏螺旋体是莱姆型疏螺旋体病的病原体，它很好地适应了它的多重扁虱和 哺乳动物的宿主，尽管极简的基因组使它失去了许多功能，但它已经高度进化 有效的机制来躲避不同的宿主防御。作为在这些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。伯格多费氏杆菌 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发现，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，我们已经确定了30多个新基因，这些基因似乎与伯氏杆菌抗药性有关。 ROS或RNS。通过将转座子文库暴露在ROS或RNS胁迫下来鉴定基因 然后进行测序(TN-seq)，以精确确定单个突变体的适合度。已识别的基因 用单个突变体和互补突变体进行了验证。基因的突变被鉴定为具有 检测了它们在ROS和/或RNS抗性中的作用及其对其自然寄主和小鼠的感染性。我们 鉴定出多个对小鼠或扁虱有感染力缺陷的突变体。 在这项建议中，我们试图了解伯氏杆菌逃避氧化的独特机制。 和亚硝化性杀戮。我们提出了以下三个具体目标。首先，在目标1中，我们将评估 伯氏杆菌ROS/RNS反应蛋白在哺乳动物感染过程中的存活。两者都使用 突变细菌和缺乏ROS/RNS防御的特定成分的基因敲除小鼠，我们将解剖 不同时间点的特定基因在感染周期中的作用。我们将使用活体成像，rna-seq 以及定量RT-PCR，以确定单个基因的作用。接下来，在目标2中，我们将评估 伯氏杆菌的ROS/RNS反应蛋白在其扁虱宿主中的生存策略与目标1相似。 最后，在目标3中，我们将确定候选蛋白如何调节对ROS的抗性。为了实现这一目标，我们将 测试几种靶向蛋白在ROS/RNS解毒中的预测活性。 这项研究代表了第一个全球评估伯氏杆菌如何对抗ROS/RNS的遗传筛查。 鉴于这种反应在宿主先天免疫中的重要性，这些偏侧螺旋体的特征 基因/蛋白质将为伯氏杆菌避免清除和持续存在的能力提供重要的洞察。 在自然界中它们占据宿主的时间较长。对生物体通过哪些机制 能够逃脱免疫介导的杀戮很可能对理解人类 莱姆病。