A unique approach to identify markers for congenital syphilis and neurosyphilis

描述（由申请人提供）：广泛挑战领域-转化科学（15）具体挑战主题- 15- od (ORDR)-101*：预防、早期发现和治疗罕见疾病的试点项目。梅毒是由螺旋体，梅毒螺旋体亚种（T. pallidum）引起的一种慢性多系统疾病。螺旋体是通过性接触获得的，在世界范围内普遍存在。先天梅毒和神经梅毒的表现在西方国家已经变得罕见，但在非洲、中亚和东欧的不发达国家仍然是一个问题。先天性梅毒是毁灭性的，可导致死产、流产、低出生体重或早产。神经梅毒也在世界各地重新出现。苍白螺旋体序列是细菌中最小的基因组之一（1.1Mb），具有几个潜在的表面蛋白，可以决定螺旋体在感染过程中定植各种组织的能力。螺旋体是细胞外病原体，它们对哺乳动物细胞的粘附似乎在组织定植中起关键作用。然而，一些挑战导致了对苍白球绦虫发病机制的了解不足。(i)苍白球绦虫不能在体外生长。这限制了彻底研究这种生物的能力。（ii）遗传技术对这种螺旋体是不可行的。（iii）苍白球绦虫表现出极强的宿主特异性，人类是其唯一的天然宿主。因此，迫切需要开发新的策略，利用小动物模型系统来研究白僵菌发病机制的分子基础，特别是与先天性梅毒和神经梅毒相关的分子基础。苍白体与引起莱姆病的伯氏疏螺旋体密切相关。这两种螺旋体在结构和生理上都是相似的生物体，并共享几种同源蛋白。T. pallidum和B. burgdorferi在其宿主中表现出不同的疾病阶段，包括各种组织的播散性感染，随后是潜伏性和慢性疾病。在过去的十年中，伯氏疏螺旋体遗传工具的发展导致了对莱姆病发病机制的理解取得了重大进展。在本文中，我们将采用一种高度创新和非常规的方法，利用生物发光伯氏疏螺旋体作为替代系统来研究选定的T. pallidum分子在细胞特异性相互作用和发病机制中的作用。我们的假设是，在生物发光的伯氏疏螺旋体中表达一种或多种T. pallidum蛋白会促进特定部位的入侵和定植。侵入胎盘，可能导致先天性传播和中枢神经系统将检测到小鼠体内成像系统（IVIS 200）。下面的研究将检验这一假设。特异性目的1：检测白螺旋体蛋白在伯氏疏螺旋体中的表达和定位，并评估其在体外粘附特定哺乳动物细胞系中的作用。特异性目的2：确定表达T. pallidum蛋白后，伯氏疏螺旋体是否获得有效定殖小鼠脑和胎盘的能力，并促进莱姆病螺旋体从胎盘向产仔的先天性传播。本研究提出了一种新的生物发光伯氏疏螺旋体替代模型系统，将有助于可视化白螺旋体蛋白表达促进小鼠胎盘和神经元组织的播散定植。这种获得功能的方法将有助于填补对这种历史上不可培养的螺旋体的理解的空白。此外，迄今为止还没有确定有希望的梅毒候选疫苗。我们希望我们在伯氏疏螺旋体研究方面的经验和我们的合作伙伴dr。Sheila Lukehart和Arturo Centurion的研究将帮助我们实现我们的目标，即确定梅毒螺旋体的关键毒力因子，并确定它们作为诊断标志物和新型候选疫苗的潜力。

DESCRIPTION (provided by applicant): Broad Challenge Area-Translational Science (15) Specific Challenge Topic- 15-OD(ORDR)-101*: Pilot projects for prevention, early detection and treatment of rare diseases, Syphilis is a chronic, multisystemic disease caused by the spirochete, Treponema pallidum subspecies pallidum (T. pallidum). The spirochete is acquired by sexual contact and is prevalent worldwide. Congenital syphilis and neurosyphillis manifestation of the disease have become rare in the Western countries but they remain a problem in underdeveloped countries of Africa, Central Asia and Eastern Europe. Congenital syphilis is devastating and can result in stillbirths, abortions, low birth weights or premature births. Neurosyphillis is also re-emerging around the world. T. pallidum sequence revealed itself to be one of the smallest genomes (1.1Mb) among bacteria with a few potential surface proteins that can determine the spirochete's ability to colonize various tissues during infection. Spirochetes are extracellular pathogens and their adherence to mammalian cells appears to play a critical role in tissue colonization. However, several challenges have resulted in the poor understanding of T. pallidum pathogenesis. (i) T. pallidum cannot be grown in vitro. This limits the ability to investigate this organism thoroughly. (ii) Genetic techniques are not feasible for this spirochete. (iii) T. pallidum exhibits extreme host specificity with humans as its only natural host. Therefore, there is a desperate need to develop novel strategies to investigate the molecular basis of T. pallidum pathogenesis especially relevant to congenital syphilis and neurosyphillis using a small animal model system. T. pallidum is closely related to Borrelia burgdorferi, which causes Lyme disease. Both of these spirochetes are structurally and physiologically similar organisms and share several homologous proteins. T. pallidum and B. burgdorferi exhibit different phases of disease in their hosts, including disseminated infection of various tissues followed by latent and chronic disease. Development of genetic tools for B. burgdorferi in the past decade has led to a significant progress in the understanding of Lyme disease pathogenesis. In this proposal, we will employ a highly innovative and unconventional approach of using bioluminescent B. burgdorferi as a surrogate system to study the role of selected T. pallidum molecules in cell-specific interactions and pathogenesis. Our hypothesis is that expression of one or more T. pallidum proteins in bioluminescent B. burgdorferi will promote invasion and colonization of specific sites. Invasion of the placenta, which may lead to congenital transmission and of the central nervous system will be detectable in mice by an in vivo imaging system (IVIS 200). The following studies will test this hypothesis. Specific Aim 1: To examine the expression and localization of T. pallidum proteins in B. burgdorferi and assess their roles in adherence to specific mammalian cell lines in vitro. Specific Aim 2: To determine if B. burgdorferi acquires the ability to colonize brain and placenta of mice efficiently after expression of T. pallidum protein(s) and also promotes congenital transmission of Lyme spirochetes from placenta to the litter. Significance Development of a new bioluminescent B. burgdorferi surrogate model system in this proposal will help visualize disseminated colonization of the mouse placenta and neuronal tissues facilitated by the expression of T. pallidum proteins. This gain of function approach will help fill the gap in the understanding of this historic, uncultivable spirochete. Furthermore, there are no promising vaccine candidates identified against syphilis to date. We expect that our experience in B. burgdorferi and expertise of our collaborators, Drs. Sheila Lukehart and Arturo Centurion, in T. pallidum pathogenesis will help us achieve our objective to identify critical virulence factors of T. pallidum and determine their potential as diagnostic markers and novel vaccine candidates. PUBLIC HEALTH RELEVANCE: Syphilis is a chronic multisystemic disease and congenital syphilis and neurosyphillis manifestation of the disease are rare in the Western countries. Congenital syphilis is devastating and can result in stillbirths, abortions, low birth weights or premature births and neurosyphillis is also re-emerging around the world. Using bioluminescent bacteria as surrogate system to express uncultivable syphilis-causing spirochete proteins in this proposal will facilitate examination of colonization of mouse placenta and neuronal tissues and will also help in identification of new diagnostic markers and vaccine candidates.