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

描述(申请人提供)：伯氏疏螺旋体是莱姆病的病原体，是一种扁虱传播的细菌病原体，能够逃避宿主的免疫防御，在皮肤、心脏、关节和中枢神经系统建立慢性感染。尽管对伯氏杆菌致病机制的了解取得了重大进展，但由于缺乏遗传工具，尤其是那些适合高通量筛选的遗传工具，了解伯氏杆菌宿主免疫逃避机制和其他毒力特性的研究进展缓慢。大规模并行测序是细菌致病机理研究中发展迅速的一项技术。安德鲁·卡米利博士是这项提议的联合研究员，他在一种名为TN-SEQ的策略中将大规模平行测序与转座子突变配对。TN-SEQ包括根据选择压力筛选文库，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。陶林博士的实验室已经在伯氏杆菌中开发出第一个转座子突变体文库。这项建议将林博士实验室在转座子突变方面的专业知识与胡博士实验室在使用TN-seq和对伯氏杆菌免疫反应方面的专业知识结合起来。在我们的初步研究中，我们在不同的接种量下给小鼠注射了一个包含10个转座子突变的微型文库。我们发现，TN-seq在从关节组织中鉴定伯氏杆菌突变体方面是可靠的和可重复性的。然而，我们发现，在关节中建立特定克隆的小鼠之间存在差异，这表明在接种时可能存在显著的生存瓶颈。然而，尽管存在显著的个体差异，但对5只小鼠的平均结果导致突变的分布与大多数品系的输入非常接近。在目标1中，我们建议首先使用TN-SEQ来确定伯氏杆菌从接种地点传播的动力学，并表征从最初接种到生存的瓶颈程度。目前对疏螺旋体如何存活并传播到远端地点的种群动态知之甚少。这些研究将为以人口为基础的这些机制提供重要的见解。我们还将使用SCID和MyD88-/-小鼠来确定适应性免疫和先天免疫对瓶颈的贡献。这些研究将使我们能够优化目标2的研究设计，在那里我们将使用TN-seq在野生型、SCID和MyD88-/-小鼠中筛选整个文库。图书馆的放映将 使我们能够识别基因的插入，这些插入会导致小鼠感染的适合性相对丧失或增加，并在特定的远端部位建立感染。用于研究伯氏杆菌感染体内模型的TN-seq的开发，有可能极大地加速我们对参与疾病发病机制的基因的理解，以及机体逃避宿主免疫防御的能力。

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.