Use of massively parallel sequencing for identification of B. burgdorferi virulen

描述（由申请人提供）：伯氏疏螺旋体是莱姆病的病原体，是一种蜱传细菌病原体，能够逃避宿主免疫防御，在皮肤、心脏、关节和中枢神经系统中建立慢性感染。尽管对伯氏疏螺旋体发病机制的理解取得了重大进展，但由于缺乏遗传工具（尤其是那些适合高通量筛选的工具），了解伯氏疏螺旋体宿主免疫逃避机制和其他毒力特性的研究进展缓慢。大规模并行测序是一项快速发展的细菌发病机制研究技术。该提案的联合研究员 Andrew Camilli 博士在一项名为 Tn-seq 的策略中将大规模并行测序与转座子诱变结合起来。 Tn-seq 涉及针对选择压力筛选文库，然后对转座子的侧翼区域进行整体测序，以确定选择前后突变体的相对频率。林涛博士实验室开发了第一个伯氏疏螺旋体转座子突变体库。该提案将林博士实验室在转座子诱变方面的专业知识与胡博士实验室在 Tn-seq 的使用和伯氏疏螺旋体免疫反应方面的专业知识结合起来。在我们的初步研究中，我们以不同的接种量给小鼠注射了包含 10 个转座子突变体的迷你文库。我们发现 Tn-seq 在从关节组织中鉴定伯氏疏螺旋体突变体方面具有稳健性和可重复性。然而，我们发现小鼠之间在关节中特定克隆的建立方面存在差异，这表明接种时可能存在显着的生存瓶颈。然而，虽然存在显着的个体差异，但对 5 只小鼠的结果进行平均，得出的突变体分布与大多数菌株的输入密切匹配。在目标 1 中，我们建议使用 Tn-seq 首先确定伯氏疏螺旋体从接种部位传播的动力学，并表征初始接种后生存瓶颈的程度。目前，人们对疏螺旋体如何生存并传播到远端地点的种群动态知之甚少。这些研究将为人群基础上的这些机制提供重要的见解。我们还将使用 SCID 和 MyD88-/- 小鼠确定适应性和先天免疫对瓶颈的贡献。这些研究将使我们能够优化 Aim 2 的研究设计，我们将使用 Tn-seq 筛选野生型、SCID 和 MyD88-/- 小鼠的整个文库。图书馆的筛选将 使我们能够识别导致小鼠感染适应性相对丧失或增强以及在特定远端位点建立感染的基因插入。 Tn-seq 的开发用于研究伯氏疏螺旋体感染的体内模型，有可能大大加速我们对疾病发病机制中涉及的基因以及生物体逃避宿主免疫防御的能力的理解。

DESCRIPTION (provided by applicant): Borrelia burgdorferi, the causative agent of Lyme disease, is a tick-borne bacterial pathogen that is able to escape host immune defenses to establish chronic infections in the skin, heart, joints, and central nervous system. Despite significant advances in the understanding of B. burgdorferi pathogenesis, studies to understand mechanisms of host immune evasion and other virulence properties of B. burgdorferi have been slowed by a paucity of genetic tools-particularly those amenable to high-throughput screening. Massively parallel sequencing is a rapidly developing technology for the study of bacterial pathogenesis. Dr. Andrew Camilli, a co-investigator on this proposal, has paired massively parallel sequencing with transposon mutagenesis in a strategy called Tn-seq. Tn-seq involves screening of a library against a selective pressure and then sequencing the flanking regions to the transposon en masse, to identify the relative frequency of the mutants before and after selection. Dr. Tao Lin's laboratory has developed the first transposon mutant library in B. burgdorferi. This proposal pairs the expertise of Dr. Lin's laboratory in transposon mutagenesis with Dr. Hu's laboratory's expertise in the use of Tn-seq and in immune responses to B. burgdorferi. In our preliminary studies, we injected mice with a mini-library of 10 transposon mutants at different inoculation amounts. We found that Tn-seq is robust and reproducible in identifying B. burgdorferi mutants from joint tissue. However, we found that there is variability between mice in the establishment of specific clones in a joint suggesting that there may be a significant bottleneck to survival at inoculation. However, while there was significant individual variation, averaging of the results over 5 mice resulted in a distribution of mutants that closely matched the input for most strains. In Aim 1, we propose to use Tn-seq to first determine the kinetics of dissemination of B. burgdorferi from the inoculation site and to characterize the extent of the bottleneck to survival from the initial inoculation. Little is currently known about he population dynamics of how Borrelia survive and disseminate to distal sites. These studies will provide important insight into these mechanisms on a population basis. We will also establish the contribution of adaptive and innate immunity to the bottleneck using SCID and MyD88-/- mice. These studies will allow us to optimize the study design for Aim 2 where we will screen the entire library in wild type, SCID and MyD88-/- mice using Tn-seq. Screening of the library will allow us to identify insertions into genes that result in a relative loss or gain of fitness for infection of the mouse and establishment of infection at specific distal sites. The development of Tn-seq for use in the study of an in vivo model of B. burgdorferi infection has the potential to greatly accelerate our understanding of genes involved in the pathogenesis of disease and the ability of the organism to evade our host immune defenses.