Functional assessment of TprC/D and TprK proteins of syphilis causing spirochete, Treponema pallidum

科学摘要 据估计，梅毒在全球的流行率为3600万例，新增感染病例超过1100万例。 每年在世界各地。仅在美国，传染性梅毒的病例数量就稳定地 自2000年以来有所增加，这表明这种疾病仍然是一个公共卫生问题，特别是考虑到 会导致严重的神经和心血管后遗症。了解外周组织的生物学功能 梅毒螺旋体亚种的膜蛋白(OMP)。梅毒螺旋体是梅毒螺旋体的一种 梅毒发病机制研究面临的最大挑战。细胞外信号转运蛋白的功能研究进展 OMPS的障碍是缺乏独立的、纯的梅毒螺旋体培养系统，遗传上的难治性 病原菌，需要利用兔进行细菌繁殖，在梅毒螺旋体外缺乏OMPS 膜，以及这个细胞隔间的脆弱性。尽管有如此多的限制，承担研究 梅毒螺旋体OMPS可以大大提高我们对这些毒力因子在梅毒中的作用的了解。 发病机制，甚至为成功控制梅毒的新方法提供重要线索，例如通过 开发一种保护性疫苗。12个可能的OMPS/毒力因子属于梅毒螺旋体重复序列 (TPR)家族，这是一组高度免疫原性的蛋白，预计与密螺旋体同源 齿纹夜蛾(T.denticola)主要鞘蛋白(MSP)，一种具有孔蛋白和粘附素特性的表面毒力因子。 到目前为止，关于各种Tprs的功能的实验证据有限或没有。基于 与MSP的同源性，TPR的结构模型和我们的初步数据，我们假设几个TPR 还具有粘附素和孔蛋白转运蛋白的双重作用。梅毒螺旋体和伯氏疏螺旋体，引起 莱姆病是一种生理和结构上相关的螺旋体。与梅毒螺旋体不同，伯氏杆菌可能是 通过基因操作和转化来表达选定的Tprs，就像它们是其OM的组成部分一样。这个 我们的初步数据表明，当TprD2和TprD2和TprD2 TprK在一种非感染性和粘附性差的伯氏杆菌菌株的表面表达，这些蛋白质 增强对胶质瘤和上皮细胞的黏附，促进氨基酸和多肽的摄取。在这里，我们计划 为了进一步检验我们的假设并建立这些重要的毒力因子的功能，利用我们的井- 尝试了B.burgdorferi，以及T.denticola MSP突变体代理系统。我们会进行以下研究 为了验证我们的假设：(1)确定梅毒螺旋体TprC、D2和K蛋白是否介导对宿主的附着 通过识别每种细胞类型上的特定宿主受体/细胞外基质成分，以不同的方式区分细胞，以及 (2)确定梅毒螺旋体TprC、D2和K蛋白作为营养物质转运通道的作用。 意义：我们的研究将有助于更好地理解梅毒螺旋体黏附到 宿主成分，这是感染期间先天性传播和神经侵袭的关键步骤，并将 为今后进一步支持将这些抗原用作候选疫苗提供重要信息。

SCIENTIFIC ABSTRACT Syphilis has an estimated global prevalence of 36M cases, with more than 11M new infections occurring per year around the world. In the United States alone, the number of cases of infectious syphilis has steadily increased since 2000, indicating that this disease is still a public health concern, particularly considering that it can lead to serious neurological and cardiovascular sequelae. Understanding the biological function of the outer membrane proteins (OMPs) of the syphilis agent, Treponema pallidum subsp. pallidum (T. pallidum), is one of the greatest challenges in the study of syphilis pathogenesis. Progress towards functional characterization of OMPs is hindered by the lack of an independent, pure culture system for T. pallidum, genetic intractability of the pathogen, the need to use of rabbits for bacterial propagation, the paucity of OMPs in T. pallidum outer membrane, and the fragility of this cellular compartment. Despite so many limitations, undertaking the study of T. pallidum OMPs can greatly enhance our understanding of the role of these virulence factors in syphilis pathogenesis and even provide important clues on new approaches to successfully control syphilis, such as by developing a protective vaccine. Twelve of putative OMPs/virulence factors belong to the T. pallidum repeat (Tpr) family, which are a group of highly immunogenic proteins predicted to be homologous to the Treponema denticola (T. denticola) major sheath protein (Msp), a surface virulence factor with porin and adhesin properties. Limited or no experimental evidence is available until now regarding functions of various Tprs. Based on homology to Msp, structural models for the Tprs and our preliminary data, we hypothesize that several Tprs also have dual roles as adhesins and porin transporters. T. pallidum and Borrelia burgdorferi, which causes Lyme disease, are physiologically and structurally related spirochetes. Unlike T. pallidum, B. burgdorferi can be genetically manipulated and transformed to express selected Tprs as if they were constituents of its OM. The validity of the surrogate system to study Tprs is supported by our preliminary data showing that when TprD2 and TprK are expressed on the surface of a non-infectious and poorly adherent B. burgdorferi strain, these proteins enhance adhesion to Glioma and epithelial cells and facilitate amino acids and peptides uptake. Here, we plan to further test our hypothesis and establish function of these important virulence factors by using both our well- tried B. burgdorferi, as well as the T. denticola msp mutant surrogate systems. We will conduct following studies to test our hypothesis: (1) Determine whether T. pallidum TprC, D2, and K proteins mediate attachment to host cells in differential manner by recognizing the specific host receptors/ECM components on each cell type, and (2) Determine the role of T. pallidum TprC, D2, and K proteins as channels involved in nutrient transport. Significance: Our studies will lead to a better understanding of the molecular basis of T. pallidum adhesion to host components, which is a pivotal step in congenital transmission and neuroinvasion during infection and will provide important information to further support the use of these antigens as vaccine candidates in the future.