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，导致盆腔炎等严重疾病导致输卵管不孕。由于缺乏可行的遗传系统和使用真核细胞作为生长介质的必要性所施加的限制，关于衣原体使用的特定毒力机制的信息缺乏。衣原体蛋白的合成是衣原体包涵体建立和成熟所必需的。包涵体成熟的最早步骤之一是向核周围区域的向心迁移，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体动力蛋白。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物，绕过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，本课题的研究目标如下：1)鉴定介导微管运动的衣原体蛋白配体，2)确定参与衣原体迁移的宿主因子。目的1将着重于鉴定参与迁移的衣原体蛋白。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筛选直接结合动力蛋白亚基。蛋白质-蛋白质相互作用将使用gst融合下拉试验确认。为了筛选衣原体效应物和动力蛋白之间的间接相互作用，将对动力蛋白亚基进行免疫沉淀，以共同沉淀衣原体蛋白。目的2将侧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转运各种各样的货物。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融合体将通过共聚焦显微镜和生化分离来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。对衣原体迁移机制的解剖将有助于了解病原体感染周期的关键早期步骤。阐明衣原体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治疗靶点以及更好地理解动力蛋白依赖的微管运输。

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.