Virulence Determinants of Borrelia burgdorferi

描述(申请人提供)：伯氏疏螺旋体是北美莱姆病的病原体，由硬蜱属的硬蜱传播。它是一种高度侵袭性的螺旋体，可在人类和其他哺乳动物中引起感染和表现，持续数月至数年。感染有局部性、播散性和持续性，伯氏杆菌似乎主要通过侵袭几乎任何组织、建立长期感染和诱导感染的能力而引起皮肤、神经、心血管和关节炎的症状。 炎症反应。该细菌不产生已知的毒素，其致病机制在很大程度上尚不清楚。由于极低的转化率和极低的质粒丢失，使用低传代、感染性伯氏杆菌进行遗传学研究一直具有挑战性；因此，通过等位基因交换或其他定点突变方法研究的基因不到50个，因为它们在哺乳动物-壁虱感染循环中具有重要作用。在之前的资助期间，在伯氏杆菌B31的可转化、有感染性的克隆中产生了一个由4,479个签名标记突变(STM)转座子突变体组成的序列定义文库。用一种新的基于Luminex的高通量策略测定了该文库中克隆的质粒量。利用该文库，我们已经对434个不同基因的484个转座子突变克隆进行了小鼠感染性的STM筛选。在目标1中，我们将利用STM突变体库完成对790个突变的蛋白编码基因在C3H/HEN小鼠感染中的作用的系统分析。在这项分析中，我们将使用在前一次资助期间开发的STM Luminex Assay协议，以高通量的方式分析每组3只小鼠、5个不同组织和两个时间点的每个突变克隆。目的2的目的是确定转座子突变对伯氏巴氏杆菌感染性、持久性和在肩部硬蜱中传播的影响。STM突变体组将通过浸泡在具有不同突变和特征标签的生物混合物中感染的幼虫传播给小鼠。STM克隆的存活率将在喂食小鼠之前和之后进行分析，克隆向小鼠的传播也将通过STM Luminex试验来确定。在目标3中，目标1和目标2中的发现将用于选择突变类型，以进行详细的感染性、互补性和功能分析。这些分析将包括对涉及趋化性、运动性、营养物质运输、参与黏附和其他作用的表面蛋白、新的基因调控途径以及在STM筛选过程中发现的其他基因组的研究。STM文库还将通过BEI-Resources向任何感兴趣的研究人员提供，提供最大限度的资源共享和对疏螺旋体研究的刺激。我们预计，该项目将继续推动有助于改善莱姆疏螺旋体病的诊断、治疗和预防的新发现。

DESCRIPTION (provided by applicant): Borrelia burgdorferi is the causative agent of Lyme disease in North America and is transmitted by ticks of the genus Ixodes. It is a highly invasive spirochete that can cause infection and manifestations in humans and other mammals that persist for months to years. The infection has localized, disseminated, and persistent phases, and B. burgdorferi appears to cause dermal, neurologic, cardiovascular, and arthritic symptoms primarily though the ability to invade almost any tissue, establish long-term infection, and induce inflammatory responses. The bacterium produces no known toxins, and its mechanisms of pathogenesis are largely unknown. Genetic studies using low-passage, infectious B. burgdorferi have been challenging due to exceedingly low transformation rates and plasmid loss; as a result, fewer than 50 genes have been investigated by allelic exchange or other site-directed mutagenesis methods for their importance in the mammal-tick infectious cycle. During the prior grant period, a sequence-defined library of 4,479 signature-tagged mutagenesis (STM) transposon mutants was generated in a transformable, infectious clone of B. burgdorferi B31. The plasmid content of the clones in this library was also determined using a novel Luminex-based high throughput strategy. Using the library, we have already performed STM screening of mouse infectivity of 484 transposon mutant clones in 434 different genes. In Aim 1, we will complete the systematic analysis of the roles of the 790 mutated protein-encoding genes in the infection of C3H/HeN mice using the STM mutant library. For this analysis, we will employ the STM Luminex Assay protocol developed during the prior grant period to analyze each mutant clone in 3 mice per group, 5 different tissues, and two time points in a high throughput manner. The goal of Aim 2 is to determine the effects of the transposon mutations on infectivity, persistence and transmission of B. burgdorferi in Ixodes scapularis ticks. Groups of the STM mutants will be transmitted to mice by larval ticks infected by immersion with a mixture of organisms with different mutations and signature tags. The survival of the STM clones will be analyzed before and after feeding on mice, and transmission of the clones to the mice will also be determined by the STM Luminex Assay. In Aim 3, the findings in Aims 1 and 2 will be used to select classes of mutants for detailed infectivity, complementation, and functional analysis. These analyses will include studies of genes involved in chemotaxis, motility, nutrient transport, surface proteins involved in adherence and other roles, novel gene regulation pathways, and additional gene sets identified during the STM screening procedure. The STM library will also be made available to any interested investigator through BEI-Resources, providing maximal resource sharing and stimulation of Borrelia research. We anticipate that this project will continue to fuel new discoveries useful in improving the diagnosis, treatment, and prevention of Lyme borreliosis.