Virulence Determinants of Borrelia burgdorferi

描述（由申请方提供）：伯氏疏螺旋体是北美莱姆病的病原体，由硬蜱属蜱传播。它是一种高度侵入性的螺旋体，可引起人类和其他哺乳动物的感染和表现，持续数月至数年。感染有局限期、播散期和持续期，B期。burgdorferi似乎主要通过侵入几乎任何组织、建立长期感染并诱导 炎症反应。该细菌不产生已知的毒素，其发病机制在很大程度上是未知的。使用低传代、传染性B的遗传学研究。由于极低的转化率和质粒损失，伯氏菌的转化一直是具有挑战性的;因此，通过等位基因交换或其它定点诱变方法研究了不到50个基因在哺乳动物-蜱感染循环中的重要性。在先前的授权期间，在B的可转化的感染性克隆中产生了4，479个签名标记的诱变（STM）转座子突变体的序列限定文库。Burgdorferi B31.该文库中克隆的质粒含量也使用新的基于Luminex的高通量策略来确定。利用该文库，我们已经对434个不同基因中的484个转座子突变克隆的小鼠感染性进行了STM筛选。目的一：利用STM突变体库，系统分析790个突变蛋白编码基因在C3 H/HeN小鼠感染中的作用。对于该分析，我们将采用先前授权期间开发的STM Luminex Assay方案，以高通量方式分析每组3只小鼠、5种不同组织和两个时间点的每个突变克隆。目的2的目标是确定转座子突变对B的感染性、持久性和传播的影响。肩胛硬蜱中的伯氏硬蜱。STM突变体组将通过浸泡具有不同突变和特征标签的生物体混合物感染的蜱虫幼虫传播给小鼠。在喂食小鼠之前和之后分析STM克隆的存活率，并且还将通过STM Luminex测定来确定克隆向小鼠的传递。在目标3中，目标1和2中的发现将用于选择突变体类别，以进行详细的感染性、互补性和功能分析。这些分析将包括参与趋化性，运动性，营养物质转运，参与粘附和其他作用的表面蛋白，新的基因调控途径，以及STM筛选过程中确定的其他基因集的研究。STM库也将通过BEI-Resources提供给任何感兴趣的研究人员，提供最大的资源共享和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病诊断、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.