Borrelia lipoproteins with the cerebral microcirculation

描述（由申请人提供）：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。一般来说，血脑屏障会排除外周的肽和蛋白质进入中枢神经系统。用蛋白质和多肽药物治疗神经系统疾病的能力在很大程度上受到这种影响的阻碍。因此，通过了解一些微生物是如何成功穿过血脑屏障的，我们可能会对药物进入中枢神经系统的发展获得非常需要的见解。脑微血管内皮细胞紧密连接形成血脑屏障。病原体可能通过细胞旁、细胞外和/或特洛伊木马机制穿过血脑屏障。微生物蛋白似乎是嗜神经病原体穿越血脑屏障能力的主要决定因素。已经在革兰氏阳性和革兰氏阴性细菌中发现了通过胞吞作用促进血脑屏障渗透的各种细菌蛋白。相比之下，人们对参与血脑屏障穿越的螺旋体蛋白知之甚少。许多螺旋体，包括梅毒螺旋体和几种导致莱姆病和回归热的疏螺旋体穿过血脑屏障。我们实验室的重点是了解莱姆病和疏螺旋体的神经系统感染的发病机制，简称神经疏螺旋体病。在感染了复发热螺旋体的小鼠中，进入中枢神经系统的部位是大脑微循环。由于分为两个家族的可变主要脂蛋白（VMP's）的顺序表达，可变大蛋白（Vlp's）和可变小蛋白（Vsp's），复发热疏螺旋体以抗原变异而闻名。在持续感染具有等基因血清型turicatae的抗体缺陷小鼠时，我们发现表达Vspl （Btl）的螺旋体引起的中枢神经系统感染和炎症是表达Vsp2 （Bt2）的螺旋体的10倍，尽管Bt2螺旋体在血液中的丰度是表达Vsp2 （Bt2）的螺旋体的10倍。最近，我们发现表达Vspl的重组伯氏疏螺旋体比表达Vsp2的重组伯氏疏螺旋体更能与人脑微血管内皮细胞（BMEC）结合，并且Vspl与BMEC细胞膜结合。这些结果暗示Vspl是已知的第一个螺旋体“神经侵入”。虽然Btl在中枢神经系统的定位是轻脑膜，但在感染小鼠中存在广泛的脑小胶质瘤，表明螺旋体产物可能扩散到脑实质。本R21应用的目的是进一步研究Vspl与脑微循环的相互作用。我们的主要假设是Vspl穿过脑微血管内皮细胞屏障。本项目的具体假设是：(i) Vspl可以从循环进入中枢神经系统，（ii） Vspl远端可变回路的特异性多态性解释了其对大脑微循环的高亲和力。我们建议对这些假设进行研究，具体目的如下：确定体内Vspl是否穿过血脑屏障。2. 调查Vspl是否进入BMEC。3. 确定Vspl是否穿过BMEC屏障。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.