Characterization of Chlamydial Inclusion Migration

描述（由申请人提供）：专性细胞内细菌沙眼衣原体是世界上可预防性失明的主要原因，也是人类最常见的性传播病原体。在美国，衣原体生殖道感染的新发病例每年约为400万例，引起严重疾病，如盆腔炎，导致输卵管不孕。由于缺乏可行的遗传系统和使用真核细胞作为生长培养基的必要性所施加的限制，衣原体所采用的特定毒力机制的信息很少。衣原体蛋白质的合成是衣原体包涵体形成和成熟所必需的。包涵体成熟的最早步骤之一是向心迁移到核周区，这是一个保守的过程中所有衣原体物种的发病机制中的重要作用。其他病毒和细菌病原体的细胞内迁移模拟宿主微管运输，因为它们需要宿主马达蛋白动力蛋白及其货物结合和激活复合物动力蛋白。衣原体包涵体迁移的机制显著不同，因为衣原体合成了一种效应物，从而避免了对动力蛋白的需求。为了更好地了解衣原体细胞内迁移和包涵体成熟，本研究计划的研究目标如下：1）鉴定介导微管运动的衣原体蛋白配体，2）确定衣原体迁移中涉及的宿主因素。目的1将集中于鉴定衣原体蛋白参与迁移。选择的候选衣原体蛋白将被筛选直接结合动力蛋白亚基使用高通量哺乳动物双杂交测定。将使用GST融合下拉试验确认蛋白质-蛋白质相互作用。为了筛选衣原体效应子和动力蛋白之间的间接相互作用，将进行动力蛋白亚基的免疫沉淀以共沉淀衣原体蛋白。目的2将集中于确定衣原体迁移所利用的动力蛋白复合物的亚基组成。动力蛋白转运各种各样的货物。这种多样性依赖于一组广泛的相关蛋白质亚基。将通过共聚焦显微镜和生化分级筛选十个动力蛋白亚基的GFP融合体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。对衣原体迁移机制的剖析将有助于了解病原体感染周期中的关键早期步骤。阐明衣原体定向迁移和宿主囊泡贩运之间的差异将导致可能的治疗目标衣原体控制以及更好地了解动力蛋白依赖微管贩运。

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.