Structure and Function 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。梅毒也是细菌慢性化和免疫逃避的一个范例，但实际上对T。苍白球执行这些神秘的过程。不幸的是，关于T的功能的信息很少。苍白球膜蛋白，可能有助于螺旋体的复杂寄生策略。T.据推测，苍白球编码45种膜脂蛋白（占其基因组的4.3%）。膜脂蛋白通常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、膜完整性的稳定剂、保护性免疫靶标和促炎激动剂也具有重要意义。然而，密螺旋体脂蛋白的功能在很大程度上仍然不确定。在传统的T。pallidum的研究，我们一直在结晶的膜脂蛋白T。苍白球和推断其功能的结构决定。最先进的生物物理和生物化学技术正在被应用于证实从结构数据得出的功能。从过去的资助间隔在解决五个T的三维结构取得的重大进展。苍白球脂蛋白（Tp 32，TP 0319 [TmpC; PnrA]，Tp 34，TP 0655 [PotD]，和Tp 0956），我们的结构生物学方法代表了在T.梅毒的非典型膜生物学（密螺旋体发病机制的一个关键方面）。此外，从以前的进展，我们现在能够提出假设驱动的实验，这将使我们能够把我们的脂蛋白功能分配更好地在T。苍白球生理学和膜生物学。鉴于此，本更新建议的具体目的是（1）评估T.（2）继续克隆并在E. coli中，纯化并结晶重组T.苍白球脂蛋白，重点是解决他们的三维结构。然后，结构数据将被用来制定新的可测试的假设，关于潜在的蛋白质功能（S）;（3）进行后续的生物学，生物化学和生物物理实验，将补充功能的预测，并将它们放在T。梅毒生物学和梅毒发病机制。阐明膜脂蛋白的功能对于理解T.梅毒膜生物学特性及其与梅毒发病的关系

DESCRIPTION (provided by applicant): Syphilis, caused by the spirochetal bacterium Treponema pallidum, continues to play prominently as a sexually transmitted disease. Syphilis also is a paradigm of bacterial chronicity and immune evasion, but virtually nothing is known about how T. pallidum carries out these enigmatic processes. Unfortunately, there is a paucity of information on the functions of T. pallidum membrane proteins that likely contribute to the spirochete's complex parasitic strategy. T. pallidum is postulated to encode 45 membrane lipoproteins (4.3% of its genome). Membrane lipoproteins typically serve many important physiological roles and also have significance as virulence factors, modular components of ABC-type transporters, stabilizers of membrane integrity, protective immune targets, and proinflammatory agonists. However, the functions of the treponemal lipoproteins have remained largely undefined. In a departure from traditional T. pallidum research, we have been crystallizing the membrane lipoproteins of T. pallidum and inferring their functions from structural determinations. State-of-the-art biophysical and biochemical techniques are being applied to corroborate functions derived from structural data. From significant progress made over the past funding interval in solving the three-dimensional structures of five T. pallidum lipoproteins (Tp32, TP0319 [TmpC; PnrA], Tp34, TP0655 [PotD], and Tp0956), it is now well documented that our structural biology approach represents a successful discovery platform for exploring lipoprotein functions in the context of T. pallidum's atypical membrane biology (a key aspect of treponemal pathogenesis). Furthermore, from prior progress, we are now able to propose hypothesis-driven experiments that will allow us to place our lipoprotein functional assignments better in the context of T. pallidum physiology and membrane biology. Given this, the Specific Aims of this renewal proposal are (1) To assess the levels of expression and membrane surface localization in T. pallidum of lipoproteins whose functions are being determined; (2) To continue to clone, express in E. coli, purify, and crystallize recombinant T. pallidum lipoproteins, with emphasis on solving their three-dimensional structures. Structural data then will be used to formulate new testable hypotheses regarding potential protein function(s); and (3) To conduct follow-up biological, biochemical, and biophysical experiments that will complement functional predictions and place them in the context of T. pallidum biology and syphilis pathogenesis. Clarifying the functions of the membrane lipoproteins is essential for understanding many of the unusual aspects of T. pallidum membrane biology and its relationship to syphilis pathogenesis.