A unique approach to identify markers for congenital syphilis and neurosyphilis

描述(由申请人提供)：广泛的挑战领域-翻译科学(15)特定挑战主题-15-OD(ORDR)-101*：预防、早期发现和治疗罕见疾病的试点项目。梅毒是由梅毒螺旋体、梅毒螺旋体亚种梅毒引起的一种慢性、多系统疾病。螺旋体是通过性接触获得的，在世界各地流行。这种疾病的先天性梅毒和神经梅毒在西方国家很少见，但在非洲、中亚和东欧等不发达国家仍然是一个问题。先天性梅毒是毁灭性的，可能导致死产、流产、低出生体重或早产。神经鞘症也在世界各地重新出现。梅毒螺旋体基因是细菌中最小的基因组之一(1.1Mb)，含有一些潜在的表面蛋白，可以决定螺旋体在感染过程中在各种组织中的定植能力。螺旋体是细胞外的病原体，它们与哺乳动物细胞的黏附似乎在组织定植中起着关键作用。然而，一些挑战导致了对梅毒螺旋体致病机制的了解不足。(I)梅毒螺旋体不能在体外生长。这限制了彻底研究这种微生物的能力。(Ii)基因技术不适用于该螺旋体。(3)梅毒螺旋体表现出极强的寄主特异性，人类是其唯一的自然寄主。因此，迫切需要开发新的策略，利用小型动物模型系统来研究梅毒螺旋体致病的分子基础，特别是与先天性梅毒和神经梅毒相关的分子基础。梅毒螺旋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，并且都有几个同源蛋白。梅毒螺旋体和伯氏杆菌在寄主中表现出不同的疾病阶段，包括各种组织的播散性感染，然后是潜伏期和慢性病。在过去的十年中，伯氏杆菌基因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。在这项提案中，我们将采用一种高度创新和非传统的方法，使用生物发光伯氏杆菌作为替代系统来研究选定的梅毒螺旋体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，一个或多个梅毒螺旋体蛋白在生物发光伯氏杆菌中的表达将促进特定部位的入侵和定植。通过体内成像系统(IVIS 200)，可以在小鼠身上检测到可能导致先天性传播的胎盘入侵和中枢神经系统的入侵。接下来的研究将验证这一假设。具体目的1：检测梅毒螺旋体蛋白在伯氏杆菌中的表达和定位，并评价其在体外与特定哺乳动物细胞系黏附中的作用。特异性目的2：确定伯氏杆菌在表达梅毒螺旋体蛋白(S)后，是否获得了高效定植小鼠脑和胎盘的能力，并促进莱姆氏螺旋体从胎盘到胎盘的先天性传播。意义发展一种新的生物发光伯氏杆菌代理模型系统将有助于可视化由梅毒螺旋体蛋白的表达促进的小鼠胎盘和神经组织的播散性定植。这种获得功能的方法将有助于填补对这种历史性的、不可培养的螺旋体的理解空白。此外，到目前为止，还没有确定有希望的梅毒疫苗候选者。我们期待我们在伯氏杆菌方面的经验以及我们的合作者Sheila Lukehart博士和Arturo Centurion博士在梅毒螺旋体致病机制方面的专业知识将帮助我们实现识别梅毒螺旋体关键毒力因素并确定它们作为诊断标记和新型疫苗候选的潜力。 公共卫生相关性：梅毒是一种慢性多系统疾病，先天性梅毒和神经梅毒的表现在西方国家很少见。先天性梅毒是毁灭性的，可能导致死产、流产、低出生体重或早产，神经梅毒也在世界各地重新出现。使用生物发光细菌作为替代系统来表达不可培养的梅毒螺旋体蛋白，将有助于检查小鼠胎盘和神经元组织的定植，并有助于识别新的诊断标记和候选疫苗。

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.