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

描述（由申请方提供）：莱姆病病原体伯氏疏螺旋体是一种蜱传细菌病原体，能够逃脱宿主免疫防御，在皮肤、心脏、关节和中枢神经系统中建立慢性感染。尽管对B的理解有了重大进展。致病机制，研究了解宿主免疫逃避机制和B的其他毒力特性。由于缺乏遗传工具，特别是那些适于高通量筛选的遗传工具，使得Burgdorferi的研究进展缓慢。大规模平行测序是一种快速发展的细菌致病机制研究技术。Andrew Camilli博士是这项提议的共同研究者，他将大规模平行测序与转座子诱变配对，采用了一种名为Tn-seq的策略。Tn-seq涉及针对选择压力筛选文库，然后对转座子基因组的侧翼区域进行测序，以确定选择前后突变体的相对频率。林涛博士的实验室开发出了B中的第一个转座子突变库。burgdorferi。该提案将Lin博士实验室在转座子诱变方面的专业知识与Hu博士实验室在使用Tn-seq和对B的免疫应答方面的专业知识结合起来。burgdorferi。在我们的初步研究中，我们以不同的接种量给小鼠注射了10个转座子突变体的微型文库。我们发现Tn-seq在鉴定B方面是稳健的和可重复的。从关节组织中分离出伯氏突变体。然而，我们发现在关节中建立特定克隆的小鼠之间存在变异性，这表明接种时存活可能存在显著瓶颈。然而，虽然存在显著的个体变异，但对5只小鼠的结果进行平均，导致突变体的分布与大多数菌株的输入密切匹配。在目的1中，我们提出使用Tn-seq首先确定B的传播动力学。burgdorferi从接种部位的生长，并表征从初始接种到存活的瓶颈的程度。目前对疏螺旋体如何存活和传播到远端位点的种群动态知之甚少。这些研究将提供重要的深入了解这些机制的人口基础上。我们还将使用SCID和MyD 88-/-小鼠建立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，其中我们将使用Tn-seq在野生型、SCID和MyD 88-/-小鼠中筛选整个文库。图书馆的筛选将 使我们能够鉴定导致小鼠感染适应性相对丧失或获得以及在特定远端位点建立感染的基因插入。用于B体内模型研究的Tn-seq的开发。伯氏菌感染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我们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.