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

项目摘要 莱姆病螺旋体持续感染和免疫逃避的关键机制 Burgdorferi是VlsE表面蛋白的抗原性变异。尽管有相当数量的 存在于细菌表面的其他蛋白质，VlsE是唯一表现出持续变异的抗原 它暴露的表位。我们实验室最近的工作发现了一种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，我们已经确定了 Arp脂蛋白作为这样一种表面抗原，受益于VlsE介导的免疫保护。尽管 这一重要证据、这一系统所涉及的某些功能和机制方面仍然未知。 我们的长期目标是确定表面抗原保护的机制和整体意义。 在伯氏杆菌感染宿主期间由VlsE脂蛋白促进。此应用程序的目标是 破译VlsE介导的免疫回避中涉及的因子的功能细节，并识别任何 被VlsE屏蔽的其他伯氏杆菌表面抗原。基于已发表的研究和 初步发现，我们的中心假设是VlsE的存在建立了与Arp的蛋白质-蛋白质相互作用 和其他蛋白质在伯氏杆菌细胞表面。此外，我们假设VlsE的存在 抑制对Arp的抗体反应。提出这项研究的理由是确定 该系统的功能细节将提供设计目标下游研究所需的知识 在剖析整体机制方面。总之，这项拟议的研究与NIH的任务有关 发展基础知识，这可能有助于减轻人类疾病的负担和 残疾。 在初步发现的指导下，我们将通过追求两个具体目标来检验我们的假设：1) 确定VlsE是否与Arp和其他伯氏杆菌细胞表面蛋白直接相互作用；以及2) 确定VlsE的存在是否调节对Arp的抗体反应。在第一个目标下，一个 专门的体内交联/洗脱技术将用于检测蛋白质与蛋白质之间的相互作用 突变的VlsE和Arp，并鉴定新的VlsE结合伙伴。在第二个目标下，qRT-PCR将是 用来定量比较VlsE缺陷克隆感染宿主期间ARP的相对表达水平 到野生型对照，感染表达或缺失VlsE克隆的小鼠的抗Arp抗体效价将 用酶联免疫吸附试验进行评估。建议的工作是创新的，因为它利用了一种新的VlsE/Arp过度表达 克隆以分析潜在的VlsE-Arp相互作用，并使用Arp脂蛋白作为VlsE保护的读数 化验。总体而言，这些研究将极大地提高我们对伯氏杆菌免疫逃避的了解， 并为预防和治疗人类莱姆病提供了更有用的策略。

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.