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，作为一种性传播疾病继续发挥着重要作用。梅毒也是细菌慢性和免疫逃避的典范，但实际上，人们对苍白t菌如何进行这些神秘的过程一无所知。不幸的是，关于T. pallidum膜蛋白功能的信息缺乏，这些功能可能有助于螺旋体的复杂寄生策略。假定苍白球绦虫编码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、保护性免疫靶点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未明确。与传统的白桦尺蠖研究不同，我们已经结晶了白桦尺蠖的膜脂蛋白，并从结构决定中推断其功能。目前正在应用最先进的生物物理和生化技术来证实从结构数据得出的功能。在过去的资助周期中，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一个成功的发现平台，用于探索梅毒螺旋体非典型膜生物学背景下的脂蛋白功能（梅毒螺旋体发病机制的一个关键方面）。此外，从先前的进展来看，我们现在能够提出假设驱动的实验，这将使我们能够在T. pallidum生理学和膜生物学的背景下更好地放置脂蛋白功能分配。鉴于此，本更新提案的具体目的是：(1)评估功能已确定的脂蛋白在苍白球中的表达水平和膜表面定位；(2)继续在大肠杆菌中克隆、表达、纯化、结晶重组苍白螺旋体脂蛋白，重点解决其三维结构问题。然后，结构数据将用于制定关于潜在蛋白质功能的新的可测试假设；(3)进行后续的生物学、生化和生物物理实验，以补充功能预测，并将其置于梅毒T. pallidum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理解梅毒螺旋体膜生物学的许多不寻常方面及其与梅毒发病机制的关系至关重要。公共卫生相关性：梅毒在美国仍然是一种重要的性传播疾病。由于无法在实验室培养梅毒螺旋体（Treponema pallidum），了解梅毒发病机制的复杂性受到了阻碍。本项目旨在利用分子生物学和结构生物学来确定生物体关键膜（脂）蛋白的结构和功能。这些膜蛋白可能在人类感染期间维持苍白t发挥战略作用。因此，我们的研究有可能阐明梅毒发病机制的许多新方面的分子基础，从而为阻止感染提供新的潜在途径。

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. PUBLIC HEALTH RELEVANCE: Syphilis remains an important sexually transmitted disease in the United States. Efforts to understand the complex nature of syphilis pathogenesis have been hindered by the inability to culture the etiological agent, Treponema pallidum, in the laboratory. This project seeks to use molecular biology and structural biology to determine the structures and functions of key membrane (lipo) proteins of the organism. These membrane proteins likely play strategic roles in sustaining T. pallidum during human infection. As such, our studies have the potential to elucidate the molecular bases of many new aspects of syphilis pathogenesis and, consequently, new potential avenues to thwart infection.