In vivo dual Bioluminescence Reporter System of Infectious Borrelia burgdorferi

传染性伯氏疏螺旋体的体内双生物发光报告系统

基本信息

批准号：
8497620
负责人：
Jenny A. Hyde
金额：
$ 20.53万
依托单位：
TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8497620
关键词：
Address American Antibiotics Antigens Arthritis Arthropod Vectors Arthropods Bacteria sigma factor KatF protein Bioluminescence Borrelia Borrelia burgdorferi Borrelia oxidative stress regulator Carbon Dioxide Cardiac Case Study Centers for Disease Control and Prevention (U.S.)Characteristics Chronic Codon Nucleotides Connective Tissue Cues Data Diagnosis Disease Environment Evaluation Exanthema Gene Expression Gene Expression Profile Gene Targeting Genes Genetic Transcription Goals Image Incidence Infection Infectious Agent Kinetics Laboratories Life Ligand Binding Domain Light Lipoproteins Luciferases Lyme Disease Modality Modeling Monitor Mus Needles Neurologic North America Order Spirochaetales Organism OspC protein Oxygen Pathogenesis Pathology Patients Pattern Phase Play Process Production Proteins Public Health Publishing Regulation Regulatory Pathway Reporter Reporting Role Skin Staging Study Section Surface System Techniques Technology Temperature Testing Tick-Borne Diseases Ticks Time Tissue-Specific Gene Expression Transcriptional Activation United States Virulence Vision Work base design erythema migrans flu in vivo innovation insight member pathogen promoter response success tissue tropism transmission process vector

项目摘要

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.

描述（由申请人提供）：众所周知，莱姆病的病原体伯氏疏螺旋体在感染期间在节肢动物载体和哺乳动物宿主之间移动时调节基因表达。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对于感染性至关重要，但 OspC 的确切功能尚不清楚。随后的研究表明，ospC 通过响应调节因子 (Rrp2) 进行协调调节，该响应调节因子与 RpoN 一起驱动 RpoS 的表达，然后促进 ospC 和其他感染性相关疏螺旋体基因的转录激活。然而，ospC 的激活是短暂的，因为它在感染后受到抑制。在这方面，如果ospC表达成为组成型，则螺旋体被迅速清除。尽管有这一观察结果，但 ospC 表达的动力学，即生命系统内随时间的推移的幅度和减少，尚不清楚。最近，我们使用体内成像来检测小鼠针接种后发光（即表达荧光素酶 [luc]）的感染性伯氏疏螺旋体。这种方法的优点是，随着时间的推移，可以在活体小鼠中多次观察伯氏疏螺旋体，以跟踪感染过程。考虑到该技术的敏感性，另一个潜在的应用可能是评估目标基因的表达。为了测试这个前提，我们将 ospC 启动子 (PospC) 与 luc 融合。我们的初步数据表明，ospC 在皮肤内感染过程的早期高度表达，但在感染后期显着减少，这与之前的报告一致，表明它在定植和传播后下调。这种方法的实用性现在将扩展到进一步研究 ospC 的空间表达以及通过 RpoS 与 ospC 协调调节的其他基因。为此，我们提出以下具体目标：（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； (2)确定参与Rrp2-RpoN-RpoS调控途径的基因的体内表达模式。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 调节是否高度协调或在伯氏疏螺旋体传播时发生在不同时间。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，重点关注已知参与实验小鼠感染的基因的启动子，应该提供对这些位点的层次结构和/或时间表达的重要洞察，以建立和维持传染焦点。这种令人兴奋的方法提供了一种强大的非侵入性实时方式，以时间和空间方式评估给定启动子的活性。