Decoding the interaction of the Chlamydia secreted effector CT192 with host dynactin

项目摘要 衣原体是目前尚不存在疫苗的疾病的重要原因。一个根本性的差距 我们所知道的是这种专属的细胞内寄生虫是如何建立一个特权的生态位--膜结合的 被称为“包含体”的隔间--为了在敌对的细胞内生存和复制 环境。衣原体编码一类独特的分泌型效应物，INCS(包涵膜蛋白)， 直接插入包涵膜。INC的位置非常适合于调解 包涵体和宿主，并可能是重要的衣原体细胞内的生存。这笔赠款的基础是 我们使用大规模亲和纯化/质谱法(AP-MS)进行了广泛的初步研究 全面鉴定沙眼衣原体与人类之间的蛋白质-蛋白质相互作用(PPI) 蛋白质组。结合严格的生物信息学分析，这项研究确定了约350个高可信的INC-HOST 38/58沙眼衣原体的PPI，代表了最全面的细菌-宿主相互作用之一 约会。令人感兴趣的是，CT192是一种功能未知的早期表达的INC，它表现出高水平的 与所有11个已知的dynactin亚基的可信度相互作用。这种多亚单位复合体调节活性。 真核生物的初级逆行微管(MT)--马达，Dynein。动力蛋白、动力蛋白和货物 接头蛋白在许多细胞过程中起着关键作用，包括囊泡和细胞器的运输。 MTS和将MTS系在中心体上。已知这两条通路都参与了C。 沙眼衣原体的生活周期，以及化学产生的CT192空突变体在 细胞培养。利用蛋白质组学、生物化学、细胞生物学和新开发的遗传学 策略I将检验CT192和dynactin之间的相互作用对 沙眼衣原体的胞内生活史。在目标1中，我将使用蛋白质组学和体外实验的组合 加工性分析映射CT192和dynactin之间的结合界面，并确定CT192如何 分别调节动力蛋白的活性。在目标2中，我将利用新开创的遗传策略来 在沙眼衣原体中建立CT192的靶向插入失活，并测试CT192- 在感染过程中动力蛋白的相互作用，以及确定CT192是否是小鼠的毒力因子 生殖道感染模型。结合这些目标，我将能够理解衣原体效应者是如何 通过对宿主重新编程，有助于创建独特的细胞内生态位。定义交互 CT192和dynactin在分子水平上的相互作用有助于理解dynactin的募集 和沙眼衣原体的调节，这可能为更广泛地了解动力蛋白的功能和调节提供新的见解。 适用于人类疾病。

Project Summary Chlamydia species are important causes of disease for which no vaccine exists. A fundamental gap in our knowledge is how this obligate intracellular parasite establishes a privileged niche- a membrane bound compartment referred to as the “inclusion”- in order to survive and replicate within the hostile intracellular environment. Chlamydiae encode a unique class secreted effectors, the Incs (inclusion membrane proteins), inserted directly into the inclusion membrane. Incs are ideally positioned to mediate interactions between the inclusion and the host, and are likely important for Chlamydia's intracellular survival. This grant builds on extensive preliminary studies in which we used large-scale affinity purification/mass spectrometry (AP-MS) to comprehensively identify protein-protein interactions (PPI) between all C. trachomatis Incs and the human proteome. Combined with rigorous bioinformatics analysis, this study identified ~350 high confidence Inc-host PPIs for 38/58 C. trachomatis Incs, representing one of the most comprehensive bacterial-host interactomes to date. Of high interest was the finding that CT192, an early expressed Inc of unknown function, exhibits high confidence interactions with all 11 known subunits of dynactin. This multi-subunit complex regulates the activity of the primary eukaryotic retrograde microtubule (MT)-motor, dynein. Dynactin, together with dynein and cargo adaptor proteins, plays critical roles in many cellular processes, including vesicle and organelle transport along MTs and tethering MTs to the centrosome. Both of these pathways are known to be involved in the C. trachomatis life cycle, and a chemically generated predicted CT192 null mutant exhibits a replication defect in cell culture. Using a combination of proteomic, biochemical, cell biological, and newly developed genetic strategies I will test the hypothesis that interaction between CT192 and dynactin is important for the intracellular life cycle of C. trachomatis. In Aim 1, I will use a combination of proteomics and in vitro processivity assays to map the binding interface between CT192 and dynactin and determine how CT192 regulates dynactin activity, respectively. In Aim 2, I will make use of newly pioneered genetic strategies to create a targeted insertional inactivation of CT192 in C. trachomatis and test the functional role of the CT192- dynactin interaction during infection, as well as determine whether CT192 is a virulence factor in a murine model of genital tract infection. Together these aims will allow me to understand how a Chlamydia effector contributes to the creation of a unique intracellular niche by reprogramming the host. Defining the interaction between CT192 and dynactin at the molecular level will contribute to the understanding of dynactin recruitment and regulation by C. trachomatis, and may provide new insights into dynactin function and regulation broadly applicable to human disease.