STRUCTURAL ANALYSIS OF PERIPLASMIC FLAGELLAR FILAMENT DYNAMICS OF

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 摘要 密螺旋体属是侵入性的。 它们能够穿透细胞单层（Lux 等，2002；Thomas 等，1988）和其他致密基质，部分原因在于它们独特的运动性。 它们的运动性是螺旋形或波浪形细胞体和周质鞭毛丝位置的结果（Limberger，1984；Ruby & Charon，1998）。 齿状密螺旋体是梅毒螺旋体亚种的模型。梅毒（梅毒病原体），以及与牙周炎相关的可培养和不可培养的口腔螺旋体。 由于最近开发了基因靶向干扰技术，对齿齿菌进行基因操作是可行的。 T. denticola 中的基因特异性中断已经增强了我们对密螺旋体生物的生物学和发病机制的了解（Izard 等，2001；Limberger 等，1999）。 最近还开发了用于蛋白质互补和表达的穿梭载体（Chi 等人，1999；Chi 等人，2002；）、Limberger 等人（未发表数据）以及新的选择方法（Limberger 等人，未发表数据）。 T. denticola 基因组序列也可用于促进拟议的研究。 齿状密螺旋体是梅毒螺旋体亚种的模型。梅毒（梅毒病原体），以及与牙周炎相关的可培养和不可培养的口腔螺旋体。 梅毒是一种急性和慢性性传播疾病。 梅毒患者还表现出感染和传播艾滋病毒的风险增加（Quinn 等，1988；Stamm 等，1988）。在美国消除梅毒需要直接或通过模型生物更好地了解致病因子，因为梅毒无法培养（St. Louis & Wasserheit，1998）。 在美国有数百万人患有牙周病。 现在人们认识到，慢性口腔感染，例如成人牙周炎，可能会产生长期后遗症（Beck等，1996；Grau等，2004）。 密螺旋体细胞数量与牙周病严重程度之间的定量关系已得到证实（Armitage 等，1982；Moter 等，1998）。 关于密螺旋体细菌周质组织的数据不完整。 旋转鞭毛束邻近周质组织。 迄今为止，仅在使用固定剂或通过冷冻断裂后才对它们的间距进行表征。 电子断层扫描最近揭示了密螺旋体中与细胞分裂相关的细胞质丝的组织（Izard 等，2004）。 未来的工作将使用更本地化的准备技术来完成。 在培养基中冷冻的整个齿垢密螺旋体的初步图像（见下文）是有希望的。 我们可以期望这些样本的断层图像中的内部特征能够得到更好的定义。 如果将鞭毛成分用作药物靶标，所提出的结构研究将对密螺旋体的细胞生物学和细胞骨架的影响提供更详细的分析。 我们研究的总体目标是了解密螺旋体运动的分子机制。 运动性使它们能够穿透致密介质和细胞层，因此是其发病机制的一个关键方面。 第一组研究旨在通过在急冻、冷冻水合的整体上进行电子断层扫描来了解周质在其自然状态下的组织，而不使用人工诱导固定剂。 第二组研究旨在确定鞭毛缺失对周质组织的影响。第三组研究将提供细胞分隔过程中鞭毛形成和插入的动态视图。 目标#1。为了完善周质鞭毛的机械和动态组织模型，将从细胞片段的 3D 重建中获得细胞结构的测量结果 了解周质和鞭毛结构将为检验与细胞运动相关的机制事件相关的假设提供机会。多个鞭毛在周质内高速旋转，它们的空间组织尚未被破译。 快速冻结将提供运动的“快照”。 目标#2。为了补充该模型，将观察鞭毛损失的超微结构效应 缺乏鞭毛的突变菌株存在结构和生化变化。 断层扫描重建将帮助我们了解鞭毛器与其他细胞特征之间的关系，包括膜完整性和肽聚糖定位。 目标#3。研究不同分裂阶段细胞分裂位点隔膜处鞭毛基体的3D空间定位 目标是识别鞭毛插入的 3D 模式。 去除外膜后获得的二维数据表明图案化。 冷冻水合整体的断层扫描应该揭示原始状态的模式。 为了补充这些分析，对鞭毛丝的进一步研究将包括与鞭毛蛋白相关的基因的敲除突变。这些包括丝的核心和外层中的蛋白质，以及通过短长度糖基化途径与核心蛋白修饰相关的蛋白质。 进一步的工作将涉及鉴定与鞭毛旋转和锚定相关的蛋白质网络。 突变型和野生型密螺旋体细胞将被带到培养基中的 RVBC，并通过浸入液体乙烷中快速冷冻。 Tilt 系列将使用 400kV JEOL 4000 TEM 在液氮温度下收集，并进行零损失能量过滤。 许多倾斜系列将围绕两个正交轴收集，这会导致更加各向同性的重建。 将使用 RVBC 开发的软件完成对齐（使用上图所示的金色标记）和重建，然后进行可视化和 3D 测量。 将以类似的方式研究分离的鞭毛。 这项研究可能会引发有关细胞子结构的问题，而这些问题无法在整体安装所能获得的分辨率水平上得到解答。 在这种情况下，细胞沉淀将被高压冷冻，并使用冷冻水合切片进行电子断层扫描。 由于这些切片可以切割至 50 nm 或更薄，因此可以获得尽可能高的分辨率。 参考文献 1. 基因组研究所 (TIGR) WWW.tigr.org。 2. rmitage, G. C.、Dickinson, W. R.、Jenderseck, R. S.、Levine, S. M. 和 Chambers, D. W. (1982)。龈下螺旋体的百分比与牙周病严重程度的关系。牙周病杂志 53, 550-556。 3. Beck, J.、Garcia, R.、Heiss, G.、Vokonas, P. S. 和 Offenbacher, S. (1996)。牙周病和心血管疾病。牙周病杂志 67, 1123-1137。 4. Chi, B.、Chauhan, S. 和 Kuramitsu, H. (1999)。用于在口腔螺旋体齿垢密螺旋体中表达异源基因的系统的开发及其在梅毒密螺旋体flaA基因表达中的应用。感染免疫 67, 3653-3656。 5. Chi, B.、Limberger, R. J. 和 Kuramitsu, H. K. (2002)。齿垢密螺旋体 flgE 突变体与新型香豆霉素 A1 抗性密螺旋体穿梭载体系统的互补。感染免疫 70，2233-2237。 6. Grau, A. J.、Becher, H.、Ziegler, C. M. 及其他作者（2004 年）。牙周病是缺血性中风的危险因素。笔画 35、496-501。 7. Izard, J.、Samsonoff, W. A. 和 Limberger, R. J. (2001)。齿垢密螺旋体细胞质丝缺陷突变体具有多效性缺陷。细菌杂志 183, 1078-1084。 8. Izard, J.、McEwen, B. F.、Barnard, R. M.、Portuese, T.、Samsonoff, W. A. 和 Limberger, R. J. (2004)。密螺旋体细胞质丝的断层扫描重建揭示了新的桥接和锚定成分。摩尔微生物学 51, 609-618。 9. Limberger, R. J. (1984)。噬菌体密螺旋体的周质鞭毛。西弗吉尼亚大学，摩根敦。 10. Limberger, R. J.、Slivienski, L. L.、Izard, J. 和 Samsonoff, W. A. (1999)。齿垢密螺旋体 tap1 的插入失活会产生具有细长鞭毛钩的非运动突变体。细菌杂志 181, 3743-3750。 11. Lux, R.、Sim, J. H.、Tsai, J. P. 和 Shi, W. (2002)。齿垢密螺旋体 cheA 突变体的构建和表征。细菌杂志 184, 3130-3134。 12. Moter, A.、Hoenig, C.、Choi, B. K.、Riep, B. 和 G¿bel, U. B. (1998)。与牙周病相关的口腔密螺旋体的分子流行病学。临床微生物杂志 36, 1399-1403。 13. Quinn, T. C.、Glasser, D.、Cannon, R. O. 及其他作者（1988 年）。性传播疾病诊所患者的人类免疫缺陷病毒感染情况。新英格兰医学杂志 318, 197-203。 14. Ruby, J.D. 和 Charon, N.W. (1998)。温度和粘度对螺旋体齿垢密螺旋体运动的影响。 FEMS 微生物快报 169, 251-254。 15. 圣路易斯，M.E. 和 Wasserheit，J.N. (1998)。美国消除梅毒。科学 281, 353-354。 16. Stamm, W. E.、Handsfield, H. H.、Rompalo, A. M.、Ashley, R. L.、Roberts, P. L. 和 Corey, L. (1988)。同性恋男性生殖器溃疡病与艾滋病毒感染之间的关联。 《美国医学会杂志》260，1429-1433。 17. Thomas, D. D.、Navab, M.、Haake, D. A.、Fogelman, A. M.、Miller, J. N. 和 Lovett, M. A. (1988)。梅毒螺旋体侵入内皮细胞单层的细胞内连接处。美国国家科学院院报 8, 3608-3612。 在上一个报告期间，为了补充收集的野生型数据，我们对缺乏鞭毛丝的突变螺旋体进行了四次断层扫描重建。 在这些中，可以清楚地看到位于周质空间鞭毛丝下方细胞质中的周质丝。 再次观察野生型的断层图，也可以在那里看到周质丝。 在分裂野生型螺旋体的重建中发现了一些先前未描述的有趣特征。 由于重建的对比度良好，并且难以交互地追踪连续切片中的螺旋特征，因此尝试根据图像参数来分割细丝。 制作了间期和分裂细胞的表面渲染模型。 六种野生型和四种无鞭毛突变体螺旋体的断层扫描重建是由冷冻的整体进行的。 首次在天然、完整的细胞中观察到 T. denticola 的所有主要特征：周质空间、鞭毛、细胞质丝和基体。 特别令人感兴趣的是鞭毛周质空间的结构，以及肽聚糖层如何容纳在那里。 在分裂细胞和细胞尖端发现了有趣的新特征。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT Treponema spp. are invasive. They are able to penetrate cell monolayers (Lux et al., 2002; Thomas et al., 1988) and other dense matrices due in part to their unique motility. Their motility is a consequence of the helical or wave-shaped cell body and the periplasmic flagellar filament location (Limberger, 1984; Ruby & Charon, 1998). Treponema denticola is the model for Treponema pallidum subsp. pallidum (the agent of syphilis), as well as cultivable and non-cultivable oral spirochetes associated with periodontitis. Genetic manipulation of T. denticola is feasible, due to the recent development of a gene targeted interruption technique. The gene-specific interruption in T. denticola has already enhanced our knowledge of the biology and the pathogenesis of treponemal organisms (Izard et al., 2001; Limberger et al., 1999). Shuttle vectors, used for complementation and expression of proteins, have also been recently developed (Chi et al., 1999; Chi et al., 2002; ), Limberger et al., (unpublished data), as well as new selection methods (Limberger et al., unpublished data). The T. denticola genomic sequence is also available facilitating the proposed studies . Treponema denticola is the model for Treponema pallidum subsp. pallidum (the agent of syphilis), as well as cultivable and non-cultivable oral spirochetes associated with periodontitis. Syphilis is an acute and chronic sexually transmitted disease. Syphilitic patients also show an increased risk for the acquisition and transmission of HIV (Quinn et al., 1988; Stamm et al., 1988). Elimination of syphilis in the U.S.A. would require a better knowledge of the responsible agent, either directly or through model organisms, since it cannot be cultivated (St. Louis & Wasserheit, 1998). Periodontal diseases are experienced by millions of people in the United States. It is now recognized that chronic oral infections, such as adult periodontitis, may have long-term sequelae (Beck et al., 1996; Grau et al., 2004). A quantitative relationship between the number of Treponema cells and the severity of periodontal disease has been demonstrated (Armitage et al., 1982; Moter et al., 1998). The data on organization of the periplasm in Treponema bacteria incomplete. Rotating flagellar bundles are organized adjacent to the periplasm. Their spacing has so far been characterized only after the use of fixatives or by freeze-fracture. Electron tomography has recently brought to light the organization of the cytoplasmic filaments associated with cell division in Treponema (Izard et al., 2004). Future work will be done with a more-native preparation technique. Preliminary images of whole mounts of Treponema denticola plunge-frozen in culture medium (see below) are promising. We can expect much better definition of the internal features in tomograms of such specimens. The structural studies proposed will provide a more detailed analysis of the consequences on the cell biology and cytoskeleon of Treponema if flagellar components were to be used as drug target. The overall goal of our research is to understand the molecular mechanisms involved in Treponema motility. Motility allows them to penetrate dense media and cell layers, and thus is a critical aspect of their pathogenesis. The first set of studies aims to understand the organization of the periplasm in its native state, without the use of artifact-inducing fixatives, by means of electron tomography carried out on plunge-frozen, frozen-hydrated whole mounts. The second set of studies aims to identify the effect of the absence of flagella on periplasmic organization. The third set of studies will provide a dynamic view of flagellar formation and insertion during cell septation. Aim #1. To refine the model of mechanical and dynamic organization of the periplasmic flagella, measurements of cell structures will be obtained from 3D reconstructions of cell segments Understanding the periplasmic and flagellar architecture will provide an opportunity to test hypotheses related to the mechanistic events associated with cell motility. Multiple flagella rotate at high speed within the periplasm, and their spatial organization in action has not yet been deciphered. Rapid freezing will provide "snapshots" of the motion. Aim #2. To complement the model, the ultrastructural effect of flagella loss will be observed Structural and biochemical changes are present in mutant strains that lack flagella. Tomographic reconstructions will help us understand the relation between the flagellar apparatus and other cell features, including membrane integrity and peptidoglycan positioning. Aim #3. To study the 3D spatial positioning of flagellar basal bodies at the septum of the cell division site in various stages of division The goal is to identify a 3D pattern of flagellar insertion. Patterning is suggested by 2D data obtained after removal of the outer membrane. Tomography of frozen-hydrated whole-mounts should reveal patterns in the native state. To complement these analyses, further work on the flagella filament will include knockout mutagenesis of genes related to flagellar proteins. These include proteins in the core and outer layer of the filaments, as well as the protein associated with core protein modification by the short length glycosylation pathway. Further work would concern identification of the network of proteins associated with flagellar rotation and anchoring. Mutant and wild-type Treponema cells will be brought to the RVBC in culture medium and quick-frozen by plunging in liquid ethane. Tilt series will be collected at liquid nitrogen temperature, with zero-loss energy filtering, using the 400kV JEOL 4000 TEM. Many of the tilt series will be collected around two orthogonal axes, which results in a more isotropic reconstruction. Alignment (using gold markers as shown in the image above) and reconstruction, followed by visualization and 3-D measurement, will be done using software developed at the RVBC. Isolated flagella will be studied in a similar manner. This research may lead to questions about cellular sub-structure that cannot be answered at the level of resolution obtainable with whole-mounts. In this case, pellets of cells will be high-pressure frozen and electron tomography will be carried out using frozen-hydrated sections. Since these sections can be cut at 50 nm and thinner, the highest possible resolution can be obtained. References 1. The Institute for Genomic Research (TIGR) WWW.tigr.org. 2. rmitage, G. C., Dickinson, W. R., Jenderseck, R. S., Levine, S. M. & Chambers, D. W. (1982). Relationship between the percentage of subgingival spirochetes and the severity of periodontal disease. J Periodontol 53, 550-556. 3. Beck, J., Garcia, R., Heiss, G., Vokonas, P. S. & Offenbacher, S. (1996). Periodontal disease and cardiovascular disease. J Periodontol 67, 1123-1137. 4. Chi, B., Chauhan, S. & Kuramitsu, H. (1999). Development of a system for expressing heterologous genes in the oral spirochete Treponema denticola and its use in expression of the Treponema pallidum flaA gene. Infect Immun 67, 3653-3656. 5. Chi, B., Limberger, R. J. & Kuramitsu, H. K. (2002). Complementation of a Treponema denticola flgE mutant with a novel coumermycin A1-resistant T. denticola shuttle vector system. Infect Immun 70, 2233-2237. 6. Grau, A. J., Becher, H., Ziegler, C. M. & other authors (2004). Periodontal disease as a risk factor for ischemic stroke. Stroke 35, 496-501. 7. Izard, J., Samsonoff, W. A. & Limberger, R. J. (2001). Cytoplasmic filament-deficient mutant of Treponema denticola has pleiotropic defects. J Bacteriol 183, 1078-1084. 8. Izard, J., McEwen, B. F., Barnard, R. M., Portuese, T., Samsonoff, W. A. & Limberger, R. J. (2004). Tomographic reconstruction of treponemal cytoplasmic filaments reveals novel bridging and anchoring components. Mol Microbiol 51, 609-618. 9. Limberger, R. J. (1984).Periplasmic flagella of Treponema phagedenis. West Virginia University, Morgantown. 10. Limberger, R. J., Slivienski, L. L., Izard, J. & Samsonoff, W. A. (1999). Insertional inactivation of Treponema denticola tap1 results in a nonmotile mutant with elongated flagellar hooks. J Bacteriol 181, 3743-3750. 11. Lux, R., Sim, J. H., Tsai, J. P. & Shi, W. (2002). Construction and characterization of a cheA mutant of Treponema denticola. J Bacteriol 184, 3130-3134. 12. Moter, A., Hoenig, C., Choi, B. K., Riep, B. & G¿bel, U. B. (1998). Molecular epidemiology of oral treponemes associated with periodontal disease. J Clin Microbiol 36, 1399-1403. 13. Quinn, T. C., Glasser, D., Cannon, R. O. & other authors (1988). Human immunodeficiency virus infection among patients attending clinics for sexually transmitted diseases. N Engl J Med 318, 197-203. 14. Ruby, J. D. & Charon, N. W. (1998). Effect of temperature and viscosity on the motility of the spirochete Treponema denticola. FEMS Microbiol Lett 169, 251-254. 15. St. Louis, M. E. & Wasserheit, J. N. (1998). Elimination of syphilis in the United States. Science 281, 353-354. 16. Stamm, W. E., Handsfield, H. H., Rompalo, A. M., Ashley, R. L., Roberts, P. L. & Corey, L. (1988). The association between genital ulcer disease and acquisition of HIV infection in homosexual men. JAMA 260, 1429-1433. 17. Thomas, D. D., Navab, M., Haake, D. A., Fogelman, A. M., Miller, J. N. & Lovett, M. A. (1988). Treponema pallidum invades intracellular junctions of endothelial cell monolayers. Proc Natl Acad Sci U S A 8, 3608-3612. In the previous reporting period, to complement the wild-type data collected, we made four tomographic reconstructions of mutant spirochetes that lack flagellar filaments. In these, the periplasmic filaments, located in the cytoplasm just beneath the flagellar filaments in the periplasmic space, could clearly be seen. Looking again at the tomograms from the wild type, the periplasmic filaments could be seen there as well. Several interesting features, not previously described, were seen in reconstructions of dividing wild-type spirochetes. Because contrast in the reconstructions was good, and since it is difficult to interactively trace spiral features in serial slices, attempts were made to segment filaments based on image parameters. Surface-rendered models of interphase and dividing cells were made. Tomographic reconstructions of six wild-type and four flagella-less mutant spirochetes were made from plunge-frozen whole-mounts. For the first time, all the main features of T. denticola were seen in native, intact cells: periplasmic space, flagella, cytoplasmic filaments, and basal bodies. Of particular interest was the structure of the periplasmic space at the flagella, and how the peptidoglycan layer is accommodated there. Interesting new features were seen in dividing cells, and at the tips of the cells.