Exploratory Studies of lp17-encoded Genetic Factors Important for Tick Colonization by the Lyme Disease Spirochete

项目摘要 伯氏疏螺旋体是一种专性寄生虫，在自然界中通过复杂的循环维持 包括蜱虫和哺乳动物宿主这两种截然不同的主机类型之间的转换需要 通过基因表达的变化快速适应的能力。B。burgdorferi拥有一个分段的 基因组由单个线性染色体和23个线性和环状质粒组成。先前 研究已经提供了在一定条件下可能差异表达的许多基因的信息， 拟模仿脊椎动物或节肢动物宿主。然而，调查才刚刚开始， 阐明了lp 17对宿主和蜱媒被B定殖的重要性。burgdorferi。出版并 我们实验室的初步工作最近表明，一个小的非编码RNA和bbd 21基因编码的， lp 17在调节对宿主适应重要的基因中具有作用。尽管有这些进步， 在我们理解许多基因的作用和功能方面， 滴答矢量。我们的长期目标是识别和表征B的必要因素。burgdorferi适应 蜱虫和哺乳动物宿主环境。此应用程序的总体目标是识别lp 17-驻留 由B定殖蜱载体所需的基因。burgdorferi。根据已发表的工作和初步的 数据，该建议的中心假设是，B.布格多费里群岛 对于蜱定殖、存活和/或传播重要。拟议研究的理由是， 这些鉴定的因子将代表疫苗和/或治疗剂开发的潜在靶点 针对莱姆病病原体的人类感染，以及针对对其地方性生活至关重要的因素 周期因此，拟议的研究与NIH的使命的一部分有关， 基础知识，这将可能有助于减少人类疾病和残疾的负担。 在引用的工作和初步数据的指导下，我们的假设将通过以下方式进行检验： 具体目标和两个子目标：1）确定蜱获得所需的lp 17-驻留基因， 生存和/或传播Bb。在第一个子目标下，肩突硬蜱蜱幼虫将首先被感染 野生型或lp 17突变体B。使用新型连续流蜱虫饲养系统， 识别对蜱虫获取和跨场生存重要的lp 17遗传区域。在第二艘潜艇下面 目的是，允许感染lp 17突变体或野生型对照的蜱若虫进食给定的时间 使用我们的蜱饲养系统，之后，在隔离的中肠和唾液腺中的细菌负荷将被 通过免疫荧光分析进行评估。当应用时，预期所建议的研究结果 最终导致新的控制措施，以破坏病原体的地方性循环，并防止感染的 莱姆病螺旋体。

Project Summary Borrelia burgdorferi is an obligate parasite, and is maintained in nature through a complex cycle involving both a tick and mammalian host. The transition between these two very different host types requires the ability to rapidly adapt through changes in gene expression. B. burgdorferi possesses a segmented genome comprised of a single linear chromosome and upwards of 23 linear and circular plasmids. Previous studies have provided information on a number of genes that may be differentially expressed under conditions intended to mimic that of the vertebrate or arthropod host. However, investigation has only just begun to elucidate the importance of lp17 for colonization of the host and tick vector by B. burgdorferi. Published and preliminary work in our lab has recently shown that a small non-coding RNA and the bbd21 gene encoded on lp17 have roles in the regulation of genes important for host adaptation. Despite these advances, there remains a fundamental gap in our understanding of the role and function of many genes for adaptation to the tick vector. Our long-term goal is to identify and characterize factors necessary for B. burgdorferi adaptation to the tick and mammalian host environments. The overall objective of this application is to identify lp17-resident genes required for colonization of the tick vector by B. burgdorferi. Based on published work and preliminary data, the central hypothesis of this proposal is that a number of lp17-resident genes of B. burgdorferi are important for tick colonization, survival, and/or transmission. The rationale for the proposed research is that these identified factors will represent potential targets for the development of a vaccine and/or therapeutics against human infection by the Lyme disease pathogen, as well as targeting factors critical for its enzootic life cycle. Thus, the proposed research is relevant to that part of NIH’s mission that pertains to developing fundamental knowledge that will potentially help to reduce the burdens of human illness and disability. Guided by cited work and preliminary data, our hypothesis will be tested by pursuing the following specific aim and two sub aims: 1) Determine the requirement of lp17-resident genes for tick acquisition, survival, and/or transmission of Bb. Under the first sub aim, Ixodes scapularis tick larvae will first be infected with either wild type or lp17 mutant B. burgdorferi clones using a novel continuous flow tick feeding system to identify genetic regions of lp17 important for tick acquisition and transstadial survival. Under the second sub aim, nymphal ticks infected with either a lp17 mutant or wild type control will be allowed to feed for a given time using our tick feeding system, after which the bacterial burden in isolated midgut and salivary glands will be assessed via immunofluorescence analysis. When applied, the results from the proposed studies are expected to ultimately lead to new control measures to disrupt the pathogen’s enzootic cycle and prevent infection by the Lyme disease spirochete.