In vivo dual Bioluminescence Reporter System of Infectious Borrelia burgdorferi

描述(申请人提供)：众所周知，伯氏疏螺旋体是莱姆病的病原体，当它在节肢动物媒介和哺乳动物宿主之间移动时，在感染过程中调节基因表达。建立哺乳动物感染所需的几个基因是已知的，典型的基因是OspC。脂蛋白OspC对于哺乳动物的感染性是必不可少的，虽然一个假定的配体结合结构域对于感染性是必不可少的，但OspC的确切功能尚不清楚。后来的研究表明，OspC是通过一个反应调节因子(RRP2)与RpoN一起协调调节的，RpoN与RpoN一起驱动rpos的表达，进而促进OspC和其他与传染性相关的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。在这一点上，如果OspC的表达是结构性的，螺旋体就会迅速清除。尽管有这样的观察，但OspC表达的动力学，即一个生命系统内随时间的幅度和减少，尚不清楚。 最近，我们利用活体成像技术检测了小鼠针刺接种后的发光(即荧光素酶[Luc]表达)感染性伯氏杆菌。这种方法的优点是，随着时间的推移，伯氏杆菌可以在活着的小鼠身上多次可视化，以跟踪感染过程。考虑到这项技术的敏感性，另一个潜在的应用可能是评估靶基因的表达。为了验证这一前提，我们将OspC启动子(POSPC)与Luc融合。我们的初步数据表明，OspC在皮肤内的感染过程早期高度表达，但在感染后期显著减少，这与先前的报告一致，表明它在定植和传播后下调。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)利用双重生物发光报告系统表征疏螺旋体OSPC在体内的组织趋向性和时间生产；(2)确定RRP2-RpoN-rpos调控途径涉及的基因在体内的表达模式。在拟议的研究中，将跟踪传播感染后OspC转录的命运以及参与感染过程的基因，以确定rpos调控是高度协调的，还是发生在伯氏杆菌传播的不同时间。 将这些特定的丝状病毒启动子-LUC结构的调控模式可视化的能力，集中在已知参与实验小鼠感染的基因的启动子上，应该提供对这些基因的层级和/或时间表达的重要洞察，以建立和维持感染焦点。这一激动人心的方法提供了一种强大的非侵入性、实时的方法来以时间和空间的方式评估给定启动子的活性。

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.