Borrelia lipoproteins with the cerebral microcirculation

描述(申请人提供)：血脑屏障(BBB)动态调节蛋白质从循环到中枢神经系统(CNS)的运动。一般而言，血脑屏障排除了外周的多肽和蛋白质，使其无法大量进入中枢神经系统。这种作用在很大程度上阻碍了用蛋白质和多肽药物治疗神经疾病的能力。因此，通过了解一些微生物如何成功地穿越血脑屏障，我们可能会为发展药物进入中枢神经系统获得亟需的洞察力。脑微血管内皮细胞的紧密连接形成血脑屏障。病原体可以通过旁细胞、跨细胞和/或特洛伊木马机制跨越血脑屏障。微生物蛋白似乎是嗜神经性病原体跨越血脑屏障能力的主要决定因素。在革兰氏阳性和革兰氏阴性细菌中，已鉴定出多种有助于跨细胞穿透血脑屏障的细菌蛋白。相比之下，人们对参与血脑屏障交叉的螺旋索蛋白知之甚少。许多螺旋体，包括梅毒螺旋体和引起莱姆病和复发性发热的几种疏螺旋体，穿过血脑屏障。我们实验室的重点是了解莱姆病神经系统感染和复发热疏螺的发病机制，称为神经疏螺旋体病。在感染回归热螺旋体的小鼠中，进入中枢神经系统的部位是大脑微循环。复发发热疏螺旋体最著名的抗原变异是由于可变主要脂蛋白(VMP)的顺序表达，VMP分为两个家族，可变大蛋白(VLP)和可变小蛋白(VSP)。在持续感染抗体缺陷小鼠的过程中，我们发现表达Vsp1(Bt1)的螺旋体引起的中枢感染和炎症是表达Vsp2(Bt2)的螺旋体的10倍，尽管Bt2的血液中螺旋体的数量是Vsp2的10倍。最近我们发现，表达Vsp1的重组伯氏疏螺旋体与人脑微血管内皮细胞(BMEC)的结合比表达Vsp2的更多，并且Vsp1与BMEC细胞膜结合。这些结果表明Vspl是第一个已知的螺旋体“神经侵袭素”。虽然BTL在中枢神经系统中的定位是软脑膜，但在感染的小鼠中存在广泛的脑小胶质细胞增生，这表明螺旋体产物可能扩散到脑实质。此R21应用的目的是进一步研究Vspl与脑微循环之间的相互作用。我们的主要假设是Vspl穿过脑微血管内皮细胞屏障。这个项目的具体假设是：(I)Vspl可以从循环进入中枢神经系统，(Ii)Vspl远端可变环的特定多态解释了它对脑微循环的高度亲和力。我们建议研究这些假说的具体目的如下：1.确定Vspl是否在体内越过了BBB。2.调查Vspl是否进入BMEC。3.确定Vspl是否跨越BMEC障碍。4.确定介导Vspl与BMEC相互作用的结构特征。

DESCRIPTION (provided by applicant): The blood brain barrier (BBB) dynamically regulates the movement of proteins from the circulation to the central nervous system (CNS). In general, the BBB excludes peptides and proteins in the periphery from substantial entry into the CNS. The ability to treat neurological disorders with protein and peptide drugs is largely hindered by this effect. Therefore, by learning how some microbes successfully cross the BBB we may gain much needed insight for the development of drug delivery into the CNS. Tight junctions in cerebral microvascular endothelial cells form the BBB. Pathogens may cross the BBB through paracellular, transcellular, and/or Trojan horse mechanisms. Microbial proteins appear to be the major determinants of the ability of neurotropic pathogens to cross the BBB. Various bacterial proteins that contribute to BBB penetration by transcytosis have been identified in gram positive and gram-negative bacteria. In contrast, little is known about the spirochetal proteins involved in crossing of the BBB. Many spirochetes, including Treponema pallidum and the several Borrelia species that cause Lyme disease and relapsing fever cross the BBB. The focus of our laboratory is to understand the pathogenesis of neurological infection with Lyme disease and relapsing fever borrelias, referred to as neuroborreliosis. In mice infected with the relapsing fever spirochete Borrelia turicatae the site of entry into the CNS is the cerebral microcirculation. Relapsing fever borrelias are best known for antigenic variation due to sequential expression of variable major lipoproteins (VMP's) divided in two families, Variable Large Proteins (Vlp's) and Variable Small Proteins (Vsp's). During persistent infection of antibody-deficient mice with isogenic serotypes of B. turicatae we found that spirochetes expressing Vspl (Btl) caused 10 times more CNS infection and inflammation than spirochetes expressing Vsp2 (Bt2) even though Bt2 spirochetes were 10 times more abundant in blood. More recently we found that recombinant Borrelia burgdorferi expressing Vspl bind to human brain microvascular endothelial cells (BMEC) more than if Vsp2 is expressed, and that Vspl binds to BMEC cell membranes. These results implicate Vspl as the first known spirochetal "neuroinvasin". Although the localization of Btl in the CNS is leptomeningeal, there is widespread cerebral microgliosis in infected mice suggesting that spirochetal products may diffuse into the brain parenchyma. The goal of this R21 application is to study further the interaction between Vspl and the cerebral microcirculation. Our main assumption is that Vspl crosses brain microvascular endothelial cell barriers. The specific hypotheses for this project are that (i) Vspl can enter the CNS from the circulation, and (ii) Specific polymorphisms in the distal variable loops of Vspl explain its high affinity for the cerebral microcirculation. We propose to study these hypotheses with the following specific aims: 1. Determine whether Vspl crosses the BBB in vivo. 2. Investigate whether Vspl enters BMEC. 3. Determine whether Vspl crosses BMEC barriers. 4. Identify the structural features that mediate the interaction of Vspl with BMEC.