Functional and Mechanistic Studies of the VlsE-mediated Immune Avoidance System in the Lyme Disease Spirochete

项目摘要 莱姆病螺旋体（Borrelia）免疫逃避和持续感染的关键机制 burgdorferi，是VlsE表面蛋白的抗原变异。尽管存在大量的 除了存在于细菌表面的其他蛋白质外，VlsE是唯一表现出持续变异的抗原。 暴露的抗原表位我们实验室最近的工作已经确定了VlsE介导的免疫回避系统， 允许非VlsE表面抗原逃避宿主抗体的杀伤作用。此外，我们还发现， 阿普脂蛋白作为一种受益于VlsE介导的免疫保护的表面抗原。尽管 这一重要证据，涉及这一系统的某些功能和机制方面仍然是未知的。 我们的长期目标是确定表面抗原保护的机制和整体意义 在B感染宿主期间由VlsE脂蛋白促进。伯格多费里。本申请的目的是 破译参与VlsE介导的免疫回避的因子的功能细节，并鉴定任何 附加B。被VlsE屏蔽的伯氏螺旋体表面抗原。根据已发表的研究和 初步发现，我们的中心假设是VlsE的存在建立了与阿普的蛋白质-蛋白质相互作用 和其他蛋白质在B上。burgdorferi细胞表面。此外，我们假设VlsE的存在 其功能是抑制抗体对阿普的反应。拟议研究的理由是， 该系统的功能细节将提供设计下游研究所需的知识， 剖析整个机制总之，拟议的研究与NIH的使命有关， 发展可能有助于减轻人类疾病负担的基础知识， 残疾。 在初步研究结果的指导下，我们的假设将通过追求两个具体目标进行测试：1） 确定VlsE是否与阿普和其他B直接相互作用。burgdorferi细胞表面蛋白;和2） 确定VlsE的存在是否调节抗体对阿普的应答。在第一个目标下， 将使用专门的体内交联/洗脱技术来检测蛋白质-蛋白质之间的相互作用。 突变的VlsE和阿普，并鉴定新的VlsE结合配偶体。在第二个目标下，qRT-PCR将是 用于定量比较VlsE缺陷型克隆在宿主感染期间的相对阿普表达水平 与野生型对照相比，用表达或缺乏VlsE的克隆感染的小鼠中的抗Arp抗体滴度将 通过ELISA进行评估。本文的工作是创新性的，因为它利用了一个新的VlsE/阿普过表达器 克隆以分析潜在的VlsE-阿普相互作用，并使用阿普脂蛋白作为VlsE保护的读数 测定。总的来说，这些研究将大大提高我们对B免疫逃避的认识。burgdorferi， 并为预防和治疗人类莱姆病提供更有用的策略。

Project Summary A key mechanism for immune evasion and persistent infection by the Lyme disease spirochete, Borrelia burgdorferi, is antigenic variation of the VlsE surface protein. Despite the presence of a substantial number of additional proteins residing on the bacterial surface, VlsE is the only antigen that exhibits ongoing variation of its exposed epitopes. Recent work in our lab has identified a VlsE-mediated immune avoidance system that allows non-VlsE surface antigens to escape the killing effects of host antibodies. Moreover, we have identified the Arp lipoprotein as one such surface antigen that benefits from VlsE-mediated immune protection. Despite this important evidence, certain functional and mechanistic aspects involved in this system are still unknown. Our long-term goals are to determine the mechanism and overall implications of surface antigen protection promoted by the VlsE lipoprotein during host infection by B. burgdorferi. The objective of this application is to decipher the functional details of factors involved in VlsE-mediated immune avoidance, and identify any additional B. burgdorferi surface antigens that are shielded by VlsE. Based on published studies and preliminary findings, our central hypothesis is that VlsE exists establishes protein-protein interactions with Arp and other proteins on the B. burgdorferi cell surface. Additionally, we hypothesize that the presence of VlsE functions to dampen the antibody response to Arp. The rationale for the proposed research is that identifying the functional details of this system will provide the knowledge required to design downstream studies targeted at dissecting the overall mechanism. Together, the proposed research is relevant to NIH’s mission that pertains to developing fundamental knowledge that will potentially help to reduce the burdens of human illness and disability. Guided by preliminary findings, our hypotheses will be tested by pursuing two specific aims: 1) Determine whether VlsE directly interacts with Arp and other B. burgdorferi cell surface proteins; and 2) Determine whether the presence of VlsE modulates the antibody response to Arp. Under the first aim, a specialized in vivo crosslinking/elution technique will be used to detect protein-protein interactions between mutant VlsE and Arp, and to identify novel VlsE binding partners. Under the second aim, qRT-PCR will be utilized to quantitate the relative arp expression levels during host infection by a VlsE-deficient clone compared to a wild type control, and anti-Arp antibody titers in mice infected with clones expressing or lacking VlsE will be assessed by ELISA. The proposed work is innovative, because it utilizes a new VlsE/Arp over-expresser clone to analyze potential VlsE-Arp interactions, and uses the Arp lipoprotein as a readout for VlsE protection assays. Overall, these studies will significantly advance our knowledge of immune evasion by B. burgdorferi, and provide more useful strategies to prevent and treat Lyme disease in humans.