STRUCTURAL ANALYSIS OF PERIPLASMIC FLAGELLAR FILAMENT DYNAMICS OF TREPONEMA: SYP

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。密螺旋体属具有侵袭性。它们能够穿透细胞单层（Lux et al., 2002; Thomas et al., 1988）和其他致密基质，部分原因是它们独特的运动性。它们的运动是螺旋状或波浪状细胞体和质周鞭毛丝位置的结果（Limberger, 1984; Ruby & Charon, 1998）。密螺旋体是梅毒螺旋体亚种的模型。梅毒（梅毒的病原体），以及与牙周炎相关的可培养和不可培养的口腔螺旋体。由于最近发展了一种基因靶向中断技术，对齿齿苋进行基因操作是可行的。denticola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认识（Izard等，2001；Limberger等，1999）。用于蛋白质互补和表达的穿梭载体最近也得到了发展（Chi et al., 1999; Chi et al., 2002;）Limberger et al.（未发表的数据），以及新的选择方法（Limberger et al.，未发表的数据）。denticola的基因组序列也可用于促进拟议的研究。密螺旋体是梅毒螺旋体亚种的模型。梅毒（梅毒的病原体），以及与牙周炎相关的可培养和不可培养的口腔螺旋体。梅毒是一种急慢性性传播疾病。梅毒患者感染和传播艾滋病毒的风险也增加（Quinn et al., 1988; Stamm et al., 1988）。在美国消灭梅毒需要更好地了解致病因子，无论是直接的还是通过模式生物，因为它不能被培养（St. Louis & Wasserheit, 1998）。在美国，数百万人患有牙周病。现在已经认识到，慢性口腔感染，如成人牙周炎，可能有长期的后遗症（Beck et al., 1996; Grau et al., 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 et al., 1982; Moter et al., 1998）。密螺旋体细菌外周质组织的资料不完整。旋转的鞭毛束与周质相邻。到目前为止，它们的间距仅在使用固定剂或冷冻断裂后才被表征。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 et al., 2004）。未来的工作将采用更原生的准备技术。在培养基中冷冻整株密螺旋体的初步图像（见下文）很有希望。我们可以期望在这些标本的断层扫描中更好地定义内部特征。如果鞭毛成分被用作药物靶点，所提出的结构研究将提供对密螺旋体细胞生物学和细胞骨架的后果的更详细的分析。我们研究的总体目标是了解参与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。第一组研究的目的是在不使用伪影诱导固定剂的情况下，通过电子断层扫描在浸入式冷冻、冷冻水合的整个载体上进行，了解其天然状态下的周质组织。第二组研究旨在确定鞭毛缺失对质周组织的影响。第三组研究将提供鞭毛的形成和插入在细胞分裂的动态视图。目标# 1。为了完善细胞质周围鞭毛的机械和动态组织模型，将通过细胞片段的三维重建获得细胞结构的测量结果。了解细胞质周围和鞭毛的结构将为测试与细胞运动相关的机械事件的假设提供机会。多个鞭毛在外周质内高速旋转，它们在活动中的空间组织尚未被破译。快速冻结将提供快照？动议的。目标# 2。为了补充该模型，将观察鞭毛丢失的超微结构效应，在缺乏鞭毛的突变菌株中存在结构和生化变化。层析重建将帮助我们了解鞭毛器官和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部位间隔处鞭毛基体的三维空间定位，目的是确定鞭毛插入的三维模式。通过去除外膜后获得的二维数据显示出图案。冷冻水合整块的断层扫描应能显示原生状态下的形态。为了补充这些分析，鞭毛细丝的进一步研究将包括鞭毛蛋白相关基因的敲除突变。这些蛋白质包括位于纤维核心层和外层的蛋白质，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识别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RVBC培养液中，并通过液体乙烷快速冷冻。倾斜系列将在液氮温度下收集，采用零损耗能量滤波，使用400kV JEOL 4000 TEM。许多倾斜序列将围绕两个正交轴收集，这将导致更各向同性的重建。校准（使用上图所示的金色标记）和重建，随后是可视化和三维测量，将使用RVBC开发的软件完成。分离鞭毛将以类似的方式进行研究。这项研究可能会导致关于细胞亚结构的问题，这些问题无法在整体安装的分辨率水平上得到回答。在这种情况下，细胞颗粒将被高压冷冻，电子断层扫描将使用冷冻的水合切片进行。由于这些部分可以在50纳米或更薄的范围内切割，因此可以获得最高的分辨率。引用1。基因组研究所（TIGR） WWW.tigr.org。2. rmitage， G. C., Dickinson， W. R., Jenderseck， R. S., Levine， S. M. & Chambers， D. W.（1982）。牙龈下螺旋体百分比与牙周病严重程度的关系。[J]中国牙周病防治杂志，2003,16(5):526 - 526。3. Beck， J., Garcia， R., Heiss， G., Vokonas， P. S.和Offenbacher， S.（1996）。牙周病和心血管疾病。[J]中华牙周病杂志，2002,11(3):526 - 526。4. Chi， B, Chauhan， S.和Kuramitsu， H.（1999）。口腔密螺旋体齿状螺旋体异种基因表达系统的建立及其在苍白密螺旋体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）。齿状密螺旋体flgE突变体与新型抗库默霉素a1齿状密螺旋体穿梭载体系统的互补。感染免疫70,2233-2237。6. 格劳，a.j.，贝彻，H.，齐格勒，c.m.和其他作者（2004年）。牙周病是缺血性中风的危险因素。35,496 -501。7. Izard， J, Samsonoff， W. A.和Limberger， R. J.（2001）。密螺旋体胞质丝缺陷突变体具有多性缺陷。[J] .中国生物医学工程学报（英文版）；8. Izard， J., McEwen, b.f., Barnard， R. M., Portuese， T., Samsonoff， W. A. & Limberger， R. J.（2004）。密螺旋体细胞质细丝的层析重建揭示了新的桥接和锚定成分。中国生物医学工程学报（英文版），51(5):693 - 698。9. 林伯格，r.j.（1984）。噬菌体密螺旋体的质周鞭毛。西弗吉尼亚大学，摩根敦。Limberger, r.j., Slivienski, l.l., Izard, j.s amsonoff, w.a.（1999）。密螺旋体denticola tap1的插入失活导致具有细长鞭毛钩的不运动突变体。[J] .中国生物医学工程学报，2001,22(3):444 - 444。11. 勒克斯,R, Sim, j . H。蔡,j.p. &史,w .(2002)。密螺旋体cheA突变体的构建与鉴定。[J] .中国生物医学工程学报，2004,22(4):393 - 398。12. Moter， A., Hoenig， C., Choi， B. K., Riep， B. & G¿bel， U. B.（1998）。口腔密螺旋体与牙周病相关的分子流行病学研究。中华临床微生物学杂志，36(2):444 - 444。13. 奎因，t.c.，格拉瑟，D，坎农，r.o.和其他作者（1988）。在性传播疾病诊所就诊的病人中感染人类免疫缺陷病毒。中华医学杂志，2003,19(3):393 - 398。14. Ruby， J. D.和Charon， N. W.（1998）。温度和黏度对密螺旋体运动的影响。微生物学通报，16(2):559 - 564。15. St. Louis, m.e. & Wasserheit, j.n.（1998）。在美国消灭梅毒。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疾病与艾滋病毒感染之间的关系。中国医学杂志26,1429-1433。17. Thomas， D. D., Navab， M., Haake， D. A., Fogelman， A. M., Miller， J. N.和Lovett， M.（1988）。梅毒螺旋体侵入内皮细胞单层的细胞内连接处。中国科学：自然科学版，36(5):387 - 398。

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.