Structure and Function of Treponema pallidum Lipoproteins

描述(申请人提供)：梅毒，由梅毒螺旋体细菌引起，作为一种性传播疾病继续发挥重要作用。梅毒也是细菌慢性化和免疫逃避的典范，但关于梅毒螺旋体是如何进行这些神秘过程的，人们几乎一无所知。不幸的是，关于梅毒螺旋体膜蛋白功能的信息很少，这可能有助于螺旋体复杂的寄生策略。梅毒螺旋体编码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，也具有重要的意义，如毒力因子、ABC转运蛋白的模块化组件、膜完整性的稳定剂、保护性免疫靶标和促炎激动剂。然而，梅毒螺旋体脂蛋白的功能在很大程度上仍未确定。与传统的梅毒螺旋体研究不同，我们一直在结晶梅毒螺旋体的膜脂蛋白，并根据结构决定推测其功能。最先进的生物物理和生化技术正被应用于证实从结构数据中得出的功能。在过去的资助期间，在解决五种梅毒螺旋体脂蛋白(Tp32，TP0319[TmpC；PNRA]，Tp34，TP0655[PotD]和Tp0956)的三维结构方面取得了重大进展，现在有充分的证据表明，我们的结构生物学方法代表了在梅毒螺旋体非典型膜生物学(梅毒螺旋体致病的一个关键方面)的背景下探索脂蛋白功能的一个成功的发现平台。此外，根据先前的进展，我们现在能够提出假设驱动的实验，使我们能够在梅毒螺旋体生理学和膜生物学的背景下更好地分配脂蛋白功能。鉴于此，本次更新方案的具体目的是(1)评估功能正在确定的脂蛋白在梅毒螺旋体中的表达水平和膜表面定位；(2)继续克隆、在大肠杆菌中表达、纯化和结晶重组梅毒螺旋体脂蛋白，重点解决其三维结构。然后将使用结构数据来制定关于潜在蛋白质功能的新的可测试假说(S)；以及(3)进行后续的生物、生化和生物物理实验，以补充功能预测，并将其置于梅毒生物学和梅毒发病机制的背景下。阐明膜脂蛋白的功能对于了解梅毒螺旋体膜生物学的许多不同寻常的方面及其与梅毒发病的关系至关重要。

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.