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

项目摘要 衣原体属是目前尚无疫苗的疾病的重要病因。一个根本性的差距， 我们的知识是这种专性细胞内寄生虫如何建立一个特权生态位-一个膜结合 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效应物，Incs（包涵体膜蛋白）， 直接插入包涵体膜。Inc的理想定位是调解 包涵体和宿主，并可能是重要的衣原体的细胞内生存。这笔赠款建立在 广泛的初步研究中，我们使用大规模亲和纯化/质谱（AP-MS）， 全面鉴定所有C.沙眼衣原体和人类 蛋白质组结合严格的生物信息学分析，本研究鉴定了~350个高置信度的Inc-宿主 38/58 C的PPI。沙眼衣原体，代表了最全面的细菌-宿主相互作用组之一， 约会高度感兴趣的是发现CT 192，一种早期表达的未知功能的Inc，表现出高表达。 与所有11个已知的动力蛋白亚基的相互作用。这种多亚基复合体调节着 原核逆行微管（MT）-马达，动力蛋白。动力蛋白，连同动力蛋白和货物 衔接蛋白，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沿着 MT和将MT拴在中心体上。已知这两种途径都参与了C. 化学产生的预测CT 192无效突变体在沙眼衣原体的生命周期中表现出复制缺陷， 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的组合， 策略我将测试的假设，CT 192和dynactin之间的相互作用是重要的， 胞内生活周期沙眼在目标1中，我将使用蛋白质组学和体外 持续合成能力测定，以绘制CT 192和动力蛋白之间的结合界面，并确定CT 192 分别调节dynactin活性。在目标2中，我将利用最新开创的遗传策略， 在C中产生CT 192的靶向插入失活。沙眼和测试的功能作用的CT 192- 在感染过程中的dynactin相互作用，以及确定CT 192是否是一个毒力因子在鼠 生殖道感染模型。这些目标将使我能够理解衣原体效应子 通过对宿主进行重编程，有助于创造独特的细胞内生态位。定义交互作用 CT 192和dynactin在分子水平上的联系将有助于理解dynactin的募集 和C.沙眼，并可能提供新的见解dynactin的功能和调控广泛 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.