STRUCT ANALYSIS PERIPLASMIC FLAGELLAR FILAMENT DYNAM OF TREPONEMA: SYPHILIS & HI

摘要 密螺旋体是侵入性的。它们能够穿透细胞单层和其他致密基质，部分原因是它们独特的运动性。它们的运动是螺旋形或波状细胞体和周质鞭毛细丝位置的结果。齿密螺旋体是梅毒螺旋体亚种的模型。梅毒(梅毒的媒介)，以及与牙周炎相关的可培养和不可培养的口腔螺旋体。由于最近基因靶向阻断技术的发展，对齿纹夜蛾的遗传操作是可行的。齿状毛滴虫的基因特异性干扰已经加深了我们对梅毒螺旋体生物学和致病机制的认识。用于蛋白质互补和表达的穿梭载体也是最近开发的，Limberger等人，以及新的选择方法。此外，还提供了齿纹夜蛾的基因组序列，为拟议的研究提供了便利。 齿密螺旋体是梅毒螺旋体亚种的模型。梅毒(梅毒的媒介)，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的性传播疾病。梅毒患者也表现出感染和传播艾滋病毒的风险增加。在美国消除梅毒需要更好地了解负责的病原体，无论是直接还是通过模式生物，因为梅毒不能培养。在美国，数以百万计的人患有牙周病。现在人们认识到，慢性口腔感染，如成人牙周炎，可能会有长期的后遗症。密螺旋体细胞的数量和牙周病的严重程度之间的定量关系已经被证明。 关于密螺旋体细菌周质组织的数据不完整。旋转的鞭毛束在周质附近组织。到目前为止，只有在使用固定剂或冷冻骨折后，才能表征它们的间距。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细丝的组织。未来的工作将使用更本地化的制备技术来完成。整个齿密螺旋体在培养基中急速冷冻的初步图像是有希望的。我们可以期待在这种标本的断层图像中更好地定义内部特征。 拟议的结构研究将提供更详细的分析，如果鞭毛成分被用作药物靶标，对密螺旋体的细胞生物学和细胞角体的影响。 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的介质和细胞层，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，方法是通过对冷冻的、冷冻水化的整个坐骑进行电子断层扫描。第二组研究旨在确定无鞭毛对周质组织的影响。第三组研究将提供细胞分离过程中鞭毛形成和插入的动态视图。 目的#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重建获得细胞结构的测量 了解周质和鞭毛的结构将提供一个机会来测试与细胞运动相关的机械事件的假说。多个鞭毛在周质内高速旋转，其作用的空间组织尚未被破译。快速冻结将提供运动的快照。 目的#2.为了补充模型，将观察鞭毛丢失的超微结构效应 缺乏鞭毛的突变菌株出现了结构和生化变化。断层重建将帮助我们了解鞭毛器和其他细胞特征之间的关系，包括膜的完整性和肽聚糖的定位。 目的#3.研究不同分裂阶段鞭毛基底体在细胞分裂部位隔膜的三维空间位置 目标是确定鞭毛插入的3D模式。通过去除外膜后获得的2D数据来建议图案化。冷冻-水合整体坐骑的断层扫描应该揭示自然状态下的模式。为了补充这些分析，关于鞭毛细丝的进一步工作将包括与鞭毛蛋白相关的基因的敲除突变。这些蛋白质包括细丝的核心和外层的蛋白质，以及与通过短长度糖基化途径修饰核心蛋白质相关的蛋白质。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。

ABSTRACT Treponema spp. are invasive. They are able to penetrate cell monolayers 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. 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. Shuttle vectors, used for complementation and expression of proteins, have also been recently developed , Limberger et al., as well as new selection methods. 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. 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. 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. A quantitative relationship between the number of Treponema cells and the severity of periodontal disease has been demonstrated. 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. Future work will be done with a more-native preparation technique. Preliminary images of whole mounts of Treponema denticola plunge-frozen in culture medium 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.