Defining B. burgdorferi oxidative stress responses by next-generation sequencing

 描述(申请人提供)：莱姆病是美国最常见的媒介传播疾病，由壁虱传播的伯氏疏螺旋体引起。伯氏杆菌在环境中的维持依赖于涉及各种哺乳动物和禽类宿主的复杂的地方病循环。伯氏假单胞菌在其蜱媒和宿主中都建立了长期感染，我们对伯氏杆菌逃避宿主免疫反应并适应它所遇到的非常不同的环境的机制非常感兴趣。除了温度、pH、金属和细胞密度等因素外，氧化应激可能代表着环境线索和伯氏杆菌的选择压力。在伯氏假单胞菌中已经发现了几个重要的抗氧化性基因，主要是基于与其他生物体中特有的基因的同源性。伯氏假单胞菌的氧化应激和氧化还原反应基因与大肠杆菌等研究较多的模式生物相比较少，氧化应激和亚硝化应激的靶标也不同，提示新的基因功能和途径可能与伯氏杆菌的氧化应激反应有关。在目前的提案中，我们将使用TN-SEQ来进行无偏见的全基因组搜索，寻找与伯氏杆菌氧化应激反应有关的基因。TN-SEQ依靠高通量测序技术来量化选择压力前后转座子文库中每个突变的频率。我们将伯氏杆菌转座子文库暴露在过氧化氢、叔丁基氢过氧化氢和一氧化氮中，并将通过产生靶向缺失和互补菌株来确认筛选中鉴定的基因。然后，我们将确定这些基因在伯氏杆菌感染其自然小鼠和壁虱宿主期间的意义。我们将确定突变和补充的伯氏杆菌菌株感染野生型小鼠以及不能产生超氧化物、次氯酸或一氧化氮的小鼠的能力，从而从机制上深入了解小鼠感染期间氧化应激的重要性和时机。我们还将测试伯氏伯氏杆菌突变体感染扁虱、在若虫蜕皮中存活并传播回小鼠的能力，从而评估氧化应激反应基因在整个流行病循环中的作用。最后，我们将使用RNA-seq研究野生型和突变型Burgdorferi对氧化应激的转录反应，使我们能够整合我们在细胞内的途径和调控网络中确定的基因。这项研究的结果将使我们更好地了解伯氏杆菌的基本生物学，并可能确定治疗中断其生命周期的新靶点。

 DESCRIPTION (provided by applicant): Lyme disease is the most common vector-borne disease in the United States and is caused by the tick-borne spirochete Borrelia burgdorferi. Maintenance of B. burgdorferi in the environment depends upon a complex enzootic cycle involving a variety of mammalian and avian reservoirs. B. burgdorferi establishes long-term infections in both its tick vector and reservoir hosts, and we are broadly interested in the mechanisms by which B. burgdorferi evades host immune responses and adapts to the very different environments it encounters. In addition to factors such as temperature, pH, metals, and cell density, oxidative stress likely represents an environmental cue and selective pressure for B. burgdorferi. A handful of genes important for conferring resistance to oxidative stress have been identified in B. burgdorferi, primarily based on homology with characterized genes in other organisms. B. burgdorferi appears to have a smaller repertoire of oxidative stress and redox-responsive genes compared to well-studied model organisms such as E. coli, and the targets of oxidative and nitrosative stress also differ between these organisms, suggesting that novel gene functions and pathways may contribute to the oxidative stress response in B. burgdorferi. In the current proposal, we will use Tn-seq to conduct an unbiased, whole-genome search for genes involved in the B. burgdorferi oxidative stress response. Tn-seq relies on high-throughput sequencing technology to quantify the frequency of every mutant in a transposon library before and after a selective pressure. We will expose a B. burgdorferi transposon library to hydrogen peroxide, t-butyl hydroperoxide, and nitric oxide and will confirm the genes identified in the screen by generating targeted deletion and complementation strains. We will then determine the significance of these genes during B. burgdorferi infection of its natural mouse and tick hosts. We will determine the ability of the mutant and complemented B. burgdorferi strains to infect wild-type mice, as well as mice that are unable to produce superoxide, hypochlorous acid, or nitric oxide, thereby gaining mechanistic insight into the significance and timing of oxidative stress during murine infection. We will also test the ability of the B. burgdorferi mutants to infet ticks, to survive the nymphal molt, and to be transmitted back to mice, thereby assessing the contribution of oxidative stress response genes throughout the enzootic cycle. Finally, we will investigate transcriptomic responses to oxidative stress in both wild-type and mutant B. burgdorferi using RNA-seq, allowing us to integrate the genes we have identified within pathways and regulatory networks in the cell. The results of this study will lead us to a better understanding of the basic biology of B. burgdorferi and may also identify new targets for therapeutic interruption of its lifecycle.