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

项目概要 莱姆病螺旋体疏螺旋体免疫逃避和持续感染的关键机制 伯氏疏螺旋体是 VlsE 表面蛋白的抗原变异。尽管存在大量 由于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 其暴露的表位。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，我们还确定了 Arp 脂蛋白作为一种这样的表面抗原，受益于 VlsE 介导的免疫保护。尽管 对于这一重要证据，该系统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总体影响 在宿主被伯氏疏螺旋体感染期间，VlsE 脂蛋白会促进这种作用。该应用程序的目的是 破译 VlsE 介导的免疫回避相关因素的功能细节，并识别任何 额外的伯氏疏螺旋体表面抗原被 VlsE 屏蔽。根据已发表的研究和 初步发现，我们的中心假设是 VlsE 的存在与 Arp 建立了蛋白质-蛋白质相互作用 以及伯氏疏螺旋体细胞表面的其他蛋白质。此外，我们假设 VlsE 的存在 具有抑制 Arp 抗体反应的功能。拟议研究的基本原理是确定 该系统的功能细节将提供设计目标下游研究所需的知识 剖析整体机制。总之，拟议的研究与 NIH 的使命相关 发展可能有助于减轻人类疾病负担的基础知识 残疾。 在初步调查结果的指导下，我们的假设将通过追求两个具体目标来检验：1) 确定 VlsE 是否直接与 Arp 和其他伯氏疏螺旋体细胞表面蛋白相互作用；和 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， 专门的体内交联/洗脱技术将用于检测蛋白质之间的相互作用 突变 VlsE 和 Arp，并鉴定新的 VlsE 结合伴侣。在第二个目标下，qRT-PCR 将 用于定量比较 VlsE 缺陷克隆在宿主感染期间的相对 arp 表达水平 与野生型对照相比，感染表达或缺乏 VlsE 的克隆的小鼠中的抗 Arp 抗体滴度将 通过 ELISA 进行评估。拟议的工作具有创新性，因为它利用了新的 VlsE/Arp 过度表达器 克隆来分析潜在的 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，这些研究将显着增进我们对伯氏疏螺旋体免疫逃避的了解， 并提供更有用的策略来预防和治疗人类莱姆病。

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.