Structural Analysis of Treponema pallidum Lipoproteins

超出所提供的空间。梅毒是由螺旋体细菌梅毒螺旋体引起的，作为一种性传播疾病，它继续发挥着重要作用。梅毒的发病机制也代表了一种细菌慢性和免疫逃避的范例，但实际上对T. paltidum如何进行这些神秘的过程一无所知。更具体地说，苍白球绦虫的外包膜由细胞质和外膜组成，必须作为人类宿主体内的物理和功能界面。不幸的是，尽管苍白球绦虫基因组已经测序，但关于苍白球绦虫膜和膜相关蛋白功能的信息仍然缺乏，这些功能可能有助于螺旋体复杂的寄生策略。在已知的膜蛋白中，苍白球绦虫被认为编码约24-35种脂蛋白。其他细菌的膜脂蛋白具有许多重要的生理作用，并且作为毒力因子、abc型转运蛋白的模块化成分、保护性免疫靶点和促炎激动剂具有重要意义，可引起强大的先天免疫反应。然而，密螺旋体脂蛋白的功能仍未明确。与传统的梅毒螺旋体研究方法不同，这项拟议的研究汇集了一组密螺旋体学家、分子生物学家、蛋白质生化学家和结构生物学家，以一种新颖的方式解决这一重要的信息缺口。本方案的具体目的是：(1)在大肠杆菌中克隆和表达苍白球的脂蛋白基因，重点是将每种多肽作为非酰化（可溶性）融合蛋白大量表达；(2)将每个融合蛋白纯化至均匀性，并对蛋白的构象进行生物物理评估；(3)获得适合x射线衍射的蛋白质晶体，求解每个可结晶脂蛋白的三维结构。其他结构生物学项目的大量初步数据和进展支持该项目的及时性和可行性；许多最先进的蛋白质结构表征技术将提高整体的成功。最后，结构数据将用于制定关于脂蛋白潜在功能的新的可测试假设，为梅毒螺旋体膜生物学和梅毒发病机制的研究提供新的途径。网站性能 ======================================== 节结束 ===========================================

EXCEED THE SPACE PROVIDED. Syphilis, caused by the spirochetal bacterium Treponema pallidum, continues to play prominently as a sexually transmitted disease. Syphilis pathogenesis also represents a paradigm of bacterial chronicity and immune evasion, but virtually nothing is known about how T. paltidum carries out these enigmatic processes. More specifically, the T. pallidum outer envelope, comprised of a cytoplasmic and outer membrane, must serve as both the physical and functional interface within the human host. Unfortunately, even though the T. pallidum genome has been sequenced, there remains a scarcity of information on the functions of T. pallidum membrane and membrane-associated proteins that likely contribute to the spirochete's complex parasitic strategy. Among the putative membrane proteins, T. pallidum is postulated to encode about 24-35 lipoproteins. Membrane lipoproteins of other bacteria subserve many important physiological roles and also have importance as virulence factors, modular components of ABC-type transporters, protective immune targets, and proinflammatory agonists that evoke robust innate immune responses. However, the functions of the treponemal lipoproteins remain essentially undefined. In a departure from more traditional approaches to T. pallidum research, the proposed study brings together a group of treponematologists, molecular biologists, protein biochemists, and structural biologists to address this important information gap in a novel way. The Specific Aims of this proposal are: (1) To clone and express in E. coli the lipoprotein genes of T. pallidum, with emphasis on expressing high quantities of each polypeptide as a nonacylated (soluble) fusion protein; (2) To purify to homogeneity each fusion protein and perform biophysical assessments of protein conformation; and (3) To obtain protein crystals suitable for X-ray diffraction and solve the three-dimensional structure for each crystallizable lipoprotein. Extensive preliminary data and progress in other structural biology initiatives support the timeliness and feasibility of this project; many state-of-the-art protein structural characterization techniques will enhance overall success. Finally, structural data will be used to formulate new testable hypotheses regarding potential function(s) of the lipoproteins, new avenues of investigation for T. pallidum membrane biology and syphilis pathogenesis that are sorely needed. PERFORMANCE SITE ========================================Section End===========================================