Structural Analysis of Treponema pallidum Lipoproteins

描述（由申请人提供）：梅毒，由螺旋体细菌梅毒螺旋体引起，继续发挥突出的性传播疾病。梅毒发病机制也代表了细菌慢性化和免疫逃避的范式，但实际上对T.苍白球执行这些神秘的过程。更具体地说，T。由细胞质和外膜组成的苍白球外被膜必须作为人类宿主内的物理和功能界面。不幸的是，即使T。虽然梅毒螺旋体基因组已被测序，但关于梅毒螺旋体功能的信息仍然很少。苍白球膜和膜相关蛋白，可能有助于螺旋体的复杂寄生策略。在推测的膜蛋白中，T.假定苍白球编码约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组分、保护性免疫靶标和引起强大的先天免疫应答的促炎激动剂也具有重要性。然而，密螺旋体脂蛋白的功能仍然基本上不确定。与传统的T.苍白球研究，拟议的研究汇集了一组密螺旋体学家，分子生物学家，蛋白质生物化学家和结构生物学家，以一种新的方式解决这一重要的信息差距。本研究的具体目的是：（1）在大肠杆菌中克隆表达; coli的脂蛋白基因。pallidum，重点在于表达大量的每种多肽作为非酰化（可溶性）融合蛋白;（2）纯化每种融合蛋白至均一性并进行蛋白质构象的生物物理评估;和（3）获得适合于X射线衍射的蛋白质晶体并解析每种可结晶脂蛋白的三维结构。广泛的初步数据和其他结构生物学倡议的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成功率。最后，结构数据将被用来制定新的可验证的假设，关于潜在的功能（S）的脂蛋白，新的途径，调查T。梅毒膜生物学和梅毒发病机制是迫切需要的。

DESCRIPTION (provided by applicant): 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. pallidum 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.