STRUCTURAL ANALYSIS OF PERIPLASMIC FLAGELLAR FILAMENT DYNAMICS OF TREPONEMA: SYP

密螺旋体周质鞭毛丝动力学的结构分析：SYP

• 批准号: 7357271
• 负责人: JACQUES G. IZARD
• 金额: $ 5.08万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7357271
• 关键词: STRUCTURAL; ANALYSIS; PERIPLASMIC; FLAGELLAR; FILAMENT

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expression of proteins, have also been recently developed (Chi et al., 1999; Chi et al., 2002; ), Limberger et al., (unpublished data), as well as new selection methods (Limberger et al., unpublished data).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.

该主题项目是利用NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构是针对该中心的，这不是调查人员的机构。抽象的treponema spp。是侵入性的。他们能够穿透细胞单层（Lux等，2002； Thomas等，1988）和其他密集的物质，部分原因是它们独特的运动性。它们的运动性是螺旋形或波形细胞体和外围鞭毛细丝位置的结果（Limberger，1984； Ruby＆Charon，1998）。 treponema denticola是treponema pallidum subsp的模型。 Pallidum（梅毒的药物），以及与牙周炎相关的可栽培和不可培养的口腔螺旋体。由于基因靶向中断技术的最新发展，对牙霉菌的遗传操作是可行的。牙霉菌中的基因特异性中断已经增强了我们对生物学生物学和发病机理的了解（Izard等，2001； Limberger等，1999）。最近也开发了用于完成和表达蛋白质的航天飞机载体（Chi等，1999; Chi等，2002;），Limberger等人（未公开的数据）以及新的选择方法（Limberger等人，未公开的数据）。牙杆菌基因组序列也可支持拟议的研究。 treponema denticola是treponema pallidum subsp的模型。 Pallidum（梅毒的药物），以及与牙周炎相关的可栽培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。梅毒患者还显示出艾滋病毒获取和传播的风险增加（Quinn等，1988； Stamm等，1988）。在美国消除梅毒将需要直接或通过模型生物体对负责人的更好了解，因为它不能培养（St. Louis＆Wasserheit，1998）。在美国，数以百万计的人经历了牙周疾病。现在已经认识到，慢性口腔感染（例如成人牙周炎）可能具有长期后遗症（Beck等，1996; Grau等，2004）。已经证明了曲科细胞数量与牙周疾病的严重程度之间的定量关系（Armitage等，1982； Moter等，1998）。关于链球菌细菌中周期肿瘤组织的数据不完整的数据。旋转的鞭毛束被组织与周质相邻。到目前为止，仅在使用固定或冻结骨折后才对其间距进行表征。电子断层扫描最近揭示了与细胞分裂相关的细胞质细丝的组织（Izard等，2004）。未来的工作将通过更本地的准备技术完成。有望在培养基中（见下文）中整个treponema denticola plunge-frozen的整个山顶的初步图像（见下文）。我们可以期望在此类标本的层析成像中更好地定义内部特征。提出的结构研究将对Treponema的细胞生物学和细胞系的后果进行更详细的分析，如果将鞭毛成分用作药物靶标。我们研究的总体目的是了解链球肿运动涉及的分子机制。运动性使他们能够穿透密集的培养基和细胞层，因此是其发病机理的关键方面。第一组研究旨在通过通过在flunge，冷冻冷冻的整个山上进行的电子断层扫描来理解其本地状态下的生物膜的组织，而无需使用伪影诱导的固定剂。第二组研究旨在确定缺乏鞭毛对周质组织的影响。第三组研究将为细胞分离期间的鞭毛形成和插入提供动态视图。目标＃1。为了完善周围鞭毛的机械和动态组织的模型，将从3D重建细胞段的重建中获得细胞结构的测量，了解外围和鞭毛结构将为测试与与细胞运动相关的机械事件相关的假设提供机会。多个鞭毛在周围内高速旋转，其空间组织尚未破译。快速冷冻会提供？快照吗？运动。目标＃2。为了完成该模型，将观察到鞭毛损失的超微结构效应，并在缺乏鞭毛的突变体菌株中提出了生化变化。层析成像的重建将有助于我们了解鞭毛设备与其他细胞特征之间的关系，包括膜完整性和PepperyDoglycan定位。目标＃3。为了研究鞭毛基本体在细胞分裂部位的隔膜上的3D空间定位，在各个划分的阶段，目标是识别鞭毛插入的3D模式。除去外膜后获得的2D数据提出了图案化。冷冻水分的整体排放量的断层扫描应揭示本地状态的模式。为了完成这些分析，对鞭毛细丝的进一步研究将包括与鞭毛蛋白有关的基因的敲除诱变。这些包括细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质修饰相关的蛋白质。进一步的工作将涉及与鞭毛旋转和锚定相关的蛋白质网络的识别。突变体和野生型treponema细胞将在培养基中通过液体乙烷浸入培养基中的RVBC和快速冻结。倾斜系列将在液氮温度下以零损失的能量过滤收集，使用400kV JEOL 4000 TEM。许多倾斜系列将在两个正交轴周围收集，从而导致更多的各向同性重建。对齐方式（使用上图所示的金标记）和重建，然后进行可视化和3-D测量，将使用RVBC开发的软件进行。孤立的鞭毛将以类似的方式进行研究。这项研究可能会导致有关细胞子结构的问题，这些问题无法在整个安装中获得的分辨率水平上回答。在这种情况下，细胞将是高压冷冻的，电子断层扫描将使用冷冻水合切片进行。由于这些部分可以在50 nm且较薄的情况下切割，因此可以获得最高的分辨率。参考文献1。基因组研究研究所（TIGR）www.tigr.org。 2。Rmitage，G.C.，Dickinson，W。R.，Jenderseck，R.S.，Levine，S.M。＆Chambers，D。W.（1982）。副螺旋体的百分比与牙周疾病的严重程度之间的关系。 J Pencenontol 53，550-556。 3。Beck，J.，Garcia，R.，Heiss，G.，Vokonas，P.S。＆Offenbacher，S。（1996）。牙周疾病和心血管疾病。 J Pencenontol 67，1123-1137。 4。Chi，B.，Chauhan，S。＆Kuramitsu，H。（1999）。开发用于口服螺旋体treponema牙本质中异源基因的系统及其在表达Treponema Pallidum flaa基因中的使用。感染免疫67，3653-3656。 5。Chi，B.，Limberger，R。J.＆Kuramitsu，H。K.（2002）。 treponema denticola flge突变体与一种新型的Comermycin A1抗牙本质T.牙本质穿梭矢量系统的互补。感染免疫70，2233-2237。 6。Grau，A。J.，Becher，H.，Ziegler，C.M。＆其他作者（2004年）。牙周疾病是缺血性中风的危险因素。中风35，496-501。 7。Izard，J。，Samsonoff，W。A.＆Limberger，R。J.（2001）。 Treponema Denticola的细胞质细胞缺乏的突变体具有多效性缺陷。 J Bacteriol 183，1078-1084。 8。Izard，J.，McEwen，B。F.，Barnard，R.M.，Portuese，T.，Samsonoff，W。A.＆Limberger，R。J.（2004）。三次细胞质细胞的层析成像重建揭示了新颖的桥接和锚定成分。 Mol Microbiol 51，609-618。 9。Limberger，R。J.（1984）。毛treponema phagedenis的周质鞭毛。西弗吉尼亚大学，摩根镇。 10。Limberger，R。J.，Slivienski，L。L.，Izard，J。＆Samsonoff，W。A.（1999）。 Treponema denticola Tap1的插入失活导致具有伸长的鞭毛钩的非运动突变体。 J Bacteriol 181，3743-3750。 11。Lux，R.，Sim，J.H.，Tsai，J.P。和Shi，W。（2002）。 Treponema denticola的CHEA突变体的结构和表征。 J Bacteriol 184，3130-3134。 12。Moter，A.，Hoenig，C.，Choi，B。K.，Riep，B。＆G¿Bel，U。B.（1998）。与牙周疾病相关的口腔TREPONOMES的分子流行病学。 J Clin Microbiol 36，1399-1403。 13。Quinn，T。C.，Glasser，D.，Cannon，R。O.＆其他作者（1988）。在性传播疾病的诊所的患者中，人类免疫缺陷病毒感染。 N Engl J Med 318，197-203。 14。Ruby，J。D.＆Charon，N。W.（1998）。温度和粘度对螺旋体毛treponema牙本质的运动性的影响。 FEMS Microbiol Lett 169，251-254。 15。圣路易斯，M。E。＆Wasserheit，J.N。（1998）。消除美国的梅毒。科学281，353-354。 16。Stamm，W。E.，Handfield，H。H.，Rompalo，A。M.，Ashley，R。L.，Roberts，P。L.＆Corey，L。（1988）。生殖器溃疡疾病与同性恋男性中HIV感染的收购之间的关联。 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侵入内皮细胞单层的细胞内连接。 Proc Natl Acad Sci U S 8，3608-3612。