Structure-Function of Borrelia Surface Lipoproteins

描述（由申请人提供）：引起回归热（RF）的疏螺旋体螺旋体可通过Vsps和Vlps的抗原变异在感染宿主中持续存在，Vsps和Vlps是两个可变的免疫显性表面脂蛋白家族，因此可定义表型。Vsps和Vlps诱导T非依赖性中和IgM应答，这导致该特定血清型的补体非依赖性消除。然而，这些抗体不能防止随后的其他血清型的螺旋体血症。突出显示了另外的逃避策略，与糖胺聚糖（GAG）具有不同结合亲和力的两种VSP之一涉及将RF疏螺旋体靶向中枢神经系统（免疫优势位点）。 本提案的总体目标是通过定义VSP和VIP介导的免疫逃避和组织嗜性的结构和功能基础来深入了解这些致病过程。它们在两种机制中的重叠作用使我们最初关注Vsps。我们的初步数据表明，Vsps共享一个保守的二聚体和α-螺旋结构折叠，高变环连接的α螺旋。我们假设这些可变的和膜远端的环（i）形成VSP特异性抗体表位，并且（ii）还赋予参与组织定位的表面性质。为了显著地促进我们的研究，我们已经开发了一种新的表面展示系统，该系统允许我们在莱姆病（LD）螺旋体伯氏疏螺旋体的表面上稳定地表达和呈现功能性Vsps和Vlps。我们现在将应用这个系统来测试我们的假设，并制定了两个主要的具体目标： 1.确定Vsp蛋白的中和抗体表位：基于两个Vsp的三维结构，利用特异性IgG和新产生的IgM单克隆抗体，通过分析Vsp逃逸突变体和抗体与Vsp嵌合体和点突变体的相互作用，定位抗体表位。 2.为了确定与GAG相互作用的Vsp结构域：将检查两种Vsp的嵌合体和点突变体与纯化的GAG、神经胶质细胞和内皮细胞的结合以及嗜神经性特征。这些研究将显着增加我们对疏螺旋体感染的发病机制的理解，并揭示更多的抗原变异和免疫逃避的微生物一般的机制。此外，它们将为未来干预策略的设计提供重要线索。

DESCRIPTION (provided by applicant): Borrelia spirochetes causing relapsing fever (RF) can persist in an infected host by antigenic variation of Vsps and Vlps, two families of variable, immunodominant and thus serotype-defining surface lipoproteins. Vsps and Vlps induce a T-independent neutralizing IgM response, which leads to complement-independent elimination of that specific serotype. Yet, these antibodies do not protect from subsequent spirochetemia of other serotypes. Highlighting an additional evasion strategy, one of two Vsps with different binding affinities to glycosaminoglycans (GAGs) has been implicated in targeting RF Borrelia to the central nervous system, an immunopriviledged site. The overall objective of this proposal is to gain an in-depth understanding of these pathogenic processes by defining the structural and functional basis of Vsp- and VIp-mediated immune evasion and tissue tropism. Their demonstrated overlapping role in both mechanisms lead us to initially focus on the Vsps. Our preliminary data indicate that Vsps share a conserved dimeric and alpha-helical structural fold, with hypervariable loops connecting the alpha helices. We hypothesize that these variable and membrane-distal loops (i) form the Vsp-specific antibody epitopes, and (ii) also confer the surface properties involved in tissue localization. To significantly facilitate our studies, we have developed a novel surface display system, which allows us to stably express and present functional Vsps and Vlps on the surface of the Lyme disease (LD) spirochete Borrelia burgdorferi. We will now apply this system to test our hypothesis and have formulated two major specific aims: 1. to define the neutralizing antibody epitopes of Vsp proteins: Based on the three-dimensional structure of two Vsps and using specific IgG and newly generated IgM monoclonal antibodies, we will map antibody epitopes by analyzing Vsp escape mutants and antibody interactions with Vsp chimeras and point mutants. 2. to define the Vsp domains interacting with GAGs: Chimeras and point mutants of two Vsps will be examined for binding to purified GAGs, glial and endothelial cells as well as for neurotropic characteristics. These studies will significantly increase our understanding of the pathogenesis of Borrelia infections, and shed more light on the mechanisms of antigenic variation and immune evasion of microbes in general. Furthermore, they will yield important clues for the design of future intervention strategies.