In vivo dual Bioluminescence Reporter System of Infectious Borrelia burgdorferi

描述（由申请人提供）：已经确定莱姆病病原体伯氏疏螺旋体在节肢动物载体和哺乳动物宿主之间移动时调节感染期间的基因表达。建立哺乳动物感染所需的几个基因是已知的，其中原型基因是ospC。脂蛋白OspC是哺乳动物感染性所必需的，尽管假定的配体结合结构域是感染性所必需的，但OspC的确切功能尚不清楚。随后的研究表明，ospC通过反应调节因子（Rrp 2）协同调节，该反应调节因子与RpoN一起驱动RpoS的表达，然后RpoS促进ospC和其他感染相关疏螺旋体基因的转录激活。然而，ospC的激活是短暂的，因为它在感染后被抑制。在这方面，如果ospC表达是组成型的，螺旋体被迅速清除。尽管有这种观察，ospC表达的动力学，即，在一个生命系统内随时间的振幅和衰减是未知的。 最近，我们已经使用体内成像来检测光发射（即，荧光素酶[luc]表达）感染性B。在针接种小鼠后的Burgdorferi。这种方法的优点是B。随着时间的推移，在活小鼠中可以多次观察到伯氏菌，以跟踪感染过程。考虑到这种技术的敏感性，另一个潜在的应用可能是评估靶基因的表达。为了测试这个前提，我们将ospC启动子（PospC）融合到luc。我们的初步数据表明，ospC在皮肤内感染过程的早期高度表达，但在感染后期显著降低，与先前的报告一致，表明它在定殖和传播后下调。这种方法的实用性现在将被扩展到进一步研究空间表达或ospC以及通过RpoS与ospC协调调节的其他基因。为此，我们提出了以下具体目标：（1）利用双生物发光报告系统表征疏螺旋体ospC的体内组织向性和时间产生;和（2）确定参与Rrp 2-RpoN-RpoS调节途径的基因的体内表达模式。在所提出的研究中，将追踪播散性感染后ospC转录的命运以及感染过程中涉及的基因，以确定RpoS调控是否高度协调或发生在与B不同的时间。burgdorferi传播。 可视化特定疏螺旋体启动子-luc构建体的这些调控模式的能力，集中于已知参与实验小鼠感染的基因的启动子，应该提供对这些基因座的层次结构和/或时间表达的重要洞察，以建立和维持感染焦点。这种令人兴奋的方法提供了一个强大的非侵入性，真实的时间模式，以评估在时间和空间的方式给定的启动子的活性。

DESCRIPTION (provided by applicant): It is well established that Borrelia burgdorferi, the etiologic agent of Lyme disease, modulates gene expression during infection as it moves between an arthropod vector and mammalian hosts. Several genes required for the establishment of mammalian infection are known with the prototypical gene being ospC. The lipoprotein OspC is absolutely required for mammalian infectivity, and although a putative ligand-binding domain is essential for infectivity, the exact function of OspC is not known. Subsequent studies indicated that ospC is coordinately regulated via a response regulator (Rrp2) that, together with RpoN, drives the expression of RpoS, which then promotes the transcriptional activation of ospC and other infectivity-associated borrelial genes. However, the activation of ospC is transient as it is repressed following infection. In this regard, if ospC expression is made constitutive, the spirochetes are rapidly cleared. Despite this observation, the kinetics of ospC expression, i.e., the amplitude and diminution within a living system over time, is not known. Recently we have used in vivo imaging to detect light emitting (i.e., luciferase [luc] expressing) infectious B. burgdorferi following needle inoculation in mice. The advantage of this approach is that B. burgdorferi can be visualized numerous times in live mice over time to track the infectious process. Given the sensitivity of this technique, an additional potential applicatio might be to assess the expression of targeted genes. To test this premise, we have fused the ospC promoter (PospC) to luc. Our Preliminary Data suggests that ospC is highly expressed early in the infectious process within skin, but is significantly reduced later in the infection, consistent with prior reports indicating that it is down regulated following colonization and dissemination. The utility of this approach will now be expanded to further study the spatial expression or ospC as well as other genes that are coordinately regulated with ospC via RpoS. To this end we propose the following Specific Aims: (1) Characterize the in vivo tissue tropism and temporal production of borrelial ospC utilizing a dual bioluminescence reporter system; and (2) Determine the in vivo expression patterns of genes involved in the Rrp2-RpoN-RpoS regulatory pathway. In the proposed studies, the fate of ospC transcription will be tracked following disseminated infection as well as genes involved in the infectious process to determine if RpoS regulation is highly coordinated or occurs at differential times as B. burgdorferi disseminates. The ability to visualize these regulatory patterns of specific borrelial promoter-luc constructs, focusing on the promoters of genes known to be involved in experimental mouse infection, should provide important insight into the hierarchy and/or temporal expression of these loci to establish and maintain an infectious focus. This exciting approach provides a powerful non-invasive, real time modality to evaluate the activity of a given promoter in a temporal and spatial manner.