Characterization of Chlamydial Inclusion Migration

衣原体包涵体迁移的表征

• 批准号: 7142424
• 负责人: SCOTT S GRIESHABER
• 金额: $ 16.2万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7142424
• 关键词: Chlamydiaceae; dynein ATPase; intracellular; microtubules; proteins; role

DESCRIPTION (provided by applicant): The obligate intracellular bacterium Chlamydia trachomatis is the world's leading cause of preventable blindness and the most common sexually transmitted pathogen of humans. In the US, the incidence of new cases of chlamydial genital tract infection is approximately 4 million annually, causing severe illness such as pelvic inflammatory disease leading to tubal infertility. Because of limitations imposed by the lack of workable genetic systems and the necessity of using eukaryotic cells as a growth medium, there is a paucity of information on specific virulence mechanisms employed by Chlamydia. Chlamydial protein synthesis is required for the establishment and maturation of the chlamydial inclusion. One of the earliest steps in inclusion maturation is centripetal migration to the perinuclear region, a process that is conserved among all chlamydial species suggesting an important role in pathogenesis. Intracellular migration by other viral and bacterial pathogens mimics host microtubule trafficking in that they require both the host motor protein dynein and its cargo binding and activating complex, dynactin. The mechanism of chlamydial inclusion migration differs significantly, as Chlamydia synthesize an effector(s) that circumvents a requirement for dynactin. To achieve a greater understanding of chlamydial intracellular migration and inclusion maturation, the research conducted in this proposal has the following goals: 1) identify the chlamydial protein ligand(s) that mediates microtubule based motility, and 2) determine host factors involved in chlamydial migration. Aim 1 will focus on identifying the chlamydial protein(s) involved in migration. Selected candidate chlamydial proteins will be screened for direct binding to subunits of dynein using a high-throughput mammalian two-hybrid assay. Protein-protein interactions will be confirmed using a GST-fusion pull down assay. To screen for indirect interactions between chlamydial effectors and dynein, immunoprecipitation of dynein subunits will be conducted to co-precipitate chlamydial proteins. Aim 2 will focus on determining the subunit composition of the dynein complex utilized by Chlamydia for migration. Dynein translocates a diverse array of cargo. This versatility depends on an extensive set of associated protein subunits. GFP fusions of the ten dynein subunits will be screened for interactions with the chlamydial inclusion by confocal microscopy and biochemical fractionation. The functional role of each subunit will be determined by siRNA inhibition studies. Dissection of the mechanism used by Chlamydia for migration will result in an understanding of a critical early step in the pathogen's infectious cycle. Elucidating the differences between chlamydial directed migration and host vesicle trafficking will lead to possible therapeutic targets for chlamydial control as well as a better understanding of dynein dependent microtubule trafficking.

描述（由申请人提供）：专性细胞内衣原体沙丘瘤是世界上可预防失明和最常见的人类性传播病原体的主要原因。在美国，新的衣原体生殖道感染病例的发生率每年约400万，导致严重疾病，例如骨盆炎性疾病，导致输卵管不育。由于缺乏可行的遗传系统施加的局限性以及将真核细胞用作生长培养基的必要性，因此关于衣原体采用的特定毒力机制的信息很少。衣原体蛋白质合成是建立和成熟衣原体包容所必需的。纳入成熟的最早步骤之一是中心迁移到核周区，这一过程在所有衣原体物种中都保守，这表明在发病机理中起重要作用。其他病毒和细菌病原体的细胞内迁移模仿宿主微管运输，因为它们需要宿主运动蛋白动力蛋白及其货物结合和激活复合物，dynactin。衣原体迁移的机制有很大的不同，因为衣原体合成了构成对Dynactin的需求的效应子。为了进一步了解衣原体的细胞内迁移和包容性成熟，该提案中进行的研究具有以下目标：1）确定介导基于微管运动的衣原体蛋白配体，以及2）确定宿主涉及的宿主因素。 AIM 1将重点放在识别涉及迁移的衣原体蛋白质上。选定的候选衣原体蛋白将使用高通量哺乳动物两杂化测定法进行直接结合与动力蛋白的亚基。蛋白质 - 蛋白质相互作用将使用GST融合下拉测定法确认。为了筛选衣原体效应子与动力蛋白之间的间接相互作用，将进行对动力蛋白亚基的免疫沉淀以共凝结衣原体蛋白。 AIM 2将着重于确定衣原体用于迁移的动力蛋白复合物的亚基组成。 Dynein易位各种货物。这种多功能性取决于广泛的相关蛋白质亚基。将筛选十个动力蛋白亚基的GFP融合，以通过共聚焦显微镜和生化分馏与衣原体包容性相互作用。每个亚基的功能作用将由siRNA抑制研究确定。衣原体用于迁移的机制的解剖会导致对病原体感染周期中关键的早期步骤的了解。阐明衣原体的定向迁移和宿主囊泡运输之间的差异将导致衣原体控制的可能的治疗靶标，并更好地了解依赖性微管运输。