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)：专性细胞内沙眼衣原体是世界上导致可预防的失明的主要原因，也是人类最常见的性传播病原体。在美国，每年新发的衣原体生殖道感染病例约为400万例，导致盆腔炎等严重疾病导致输卵管性不孕。由于缺乏可行的遗传系统和使用真核细胞作为生长介质的必要性，关于衣原体所使用的特定毒力机制的信息很少。衣原体蛋白的合成是建立和成熟衣原体包涵体所必需的。包涵体成熟的最早步骤之一是向心迁移到核周区域，这一过程在所有衣原体物种中都是保守的，表明在发病机制中发挥了重要作用。其他病毒和细菌病原体的细胞内迁移模拟宿主微管运输，因为它们既需要宿主运动蛋白dynein，也需要其货物结合和激活复合体dynactin。衣原体包涵体迁移的机制有很大的不同，因为衣原体合成了一种效应器(S)，它绕过了动力蛋白的要求。为了更好地了解衣原体在细胞内的迁移和包涵体的成熟，本研究的目标如下：1)确定介导微管运动的衣原体蛋白配体(S)；2)确定参与衣原体迁移的宿主因素。目标1将重点确定参与迁移的衣原体蛋白(S)。选定的候选衣原体蛋白将使用高通量哺乳动物双杂交试验进行直接结合动力蛋白亚基的筛选。蛋白质之间的相互作用将使用GST融合下拉试验来证实。为了筛选衣原体效应物和动力蛋白之间的间接相互作用，将进行动力蛋白亚单位的免疫沉淀以共沉淀衣原体蛋白。目标2将侧重于确定衣原体用于迁移的动力蛋白复合体的亚基组成。动力蛋白可以运输各种不同的货物。这种多功能性依赖于一组广泛的相关蛋白质亚基。十个动力蛋白亚基的GFP融合将通过共聚焦显微镜和生化分级筛选与衣原体包涵体的相互作用。每个亚基的功能作用将通过siRNA抑制研究来确定。对衣原体用于迁移的机制的剖析将导致对病原体感染周期的关键早期步骤的理解。阐明衣原体定向迁移和宿主囊泡运输之间的差异，将有助于更好地理解动力蛋白依赖的微管运输，为衣原体控制提供可能的治疗靶点。