CRYO-EM TOMOGRAPHY OF FLAGELLAR FILAMENTS OF BORRELIA BURGDORFERI

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 摘要： 螺旋体是一种具有重要医学意义但却鲜为人知的细菌群。这些微生物会引起多种重要疾病，包括梅毒、莱姆病、复发性发热和钩端螺旋体病。它们还与牙周病和人类腹泻疾病有关。研究人员的目标是回答与螺旋体周质鞭毛(PFS)的结构和运动性有关的某些问题。螺旋体PFS是所有细菌鞭毛纤维中最复杂的。在PFS的表面是由一到两种FlaA蛋白组成的蛋白质鞘。核心是由三到四种蛋白质组成的聚合物，称为FLAB蛋白质。单个Pf蛋白的功能尚不清楚。 在之前与RVBC的工作中，从突变的鞭毛中分离出的PFS已经用冷冻-EM进行了研究，目前正在准备一份手稿。在这项研究中，我们希望通过比较不同的突变菌株改变运动能力来证实在完整细胞的鞭毛中的发现。这些研究尤其相关，因为鞭毛和/或运动性被认为是螺旋体和其他几种病原菌的毒力因子。 在上一次报告所述期间，对四株伯氏疏螺旋体进行了整体冷藏冷冻扫描：B31野生型、车A、车X和Fli G1。对所有四个菌株进行断层重建。查隆博士访问了资源中心，研究了结果，并在电子显微镜下观察标本，以帮助确定未来工作的最佳样本。我们发现自然细胞中鞭毛的排列与以前在化学固定的细胞中看到的不同，与先前研究的随机薄片相比，在整个细胞的断层图像中可以更好地理解运动器官的三维排列。

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: Spirochetes are a medically significant but a remarkable poorly understood group of bacteria. These organisms cause a variety of important diseases including syphilis, Lyme disease, relapsing fever, and leptospirosis. They are also implicated in periodontal disease and human diarrheal disease. The investigators aim to answer certain questions relating to the structure of spirochete periplasmic flagella (PFs) and motility. Spirochete PFs are the most complex of any bacterial flagellar filament. On the surface of the PFs is a protein sheath comprised of one to two FlaA protein species. The core is comprised of a polymer of three to four protein species referred to as FlaB proteins. The function of the individual PF proteins is not understood. In previous work with the RVBC, isolated PFs from mutant flagella were studied by cryo-EM, and a manuscript is in preparation. In this study, we wish to confirm findings in flagella from intact cells, by comparing various mutant strains having altered motility. These studies are especially relevant in that flagella and/or motility are implicated as a virulence factor for spirochetes and several other species of pathogenic bacteria. In the previous reporting period, four strains of Borrelia burgdorferi were plunge-frozen as whole mounts for cryo-tomography: B31 wild type, Che A, Che X, and Fli G1. Tomographic reconstructions of all four strains were made. Dr. Charon visited the Resource to study the results, and to observe the specimens at the electron microscope in order to help identify the optimal examples for future work. We found the arrangement flagella in native cells to differ from that seen previously in chemically-fixed cells, and the 3-D arrangement of the motile apparatus could be much better appreciated in the tomograms of whole cells, compared to the random thin sections previously studied.