Discovery of Chlamydia-host interactions

项目总结/摘要 主要的人类病原体沙眼衣原体是一种专性细胞内细菌， 在称为内含物的细胞空泡内的复制生长周期。衣原体包涵体在 建立和维持一个有利的细胞内生态位衣原体，并通过外部包涵体 披衣细胞作为这种液泡的膜，分泌大量的效应分子进入宿主细胞。身份， 大多数这些分泌蛋白的功能和宿主相互作用靶标尚不清楚，这在很大程度上是由于 与衣原体基因操纵相关的历史挑战。最近的突破， 衣原体遗传学已经消除了这些主要障碍中的一些。 利用这些进展，我们已经开发了一个遗传蛋白质组系统的C。沙眼， 选择性地标记紧邻包涵体膜的蛋白质，使得能够分离和鉴定 这些蛋白质的质谱分析。该系统允许对包裹体进行高度特异性的交互式绘图 膜蛋白质组，甚至在感染早期，因此有可能发现细菌和 在原位构成致病区室的宿主蛋白质。该提案的主要目标是： (i)确定衣原体和宿主蛋白质，其在 三个阶段的液泡生长，和（ii）扩展这种技术的发展，用于鉴定宿主 分子靶向衣原体分泌蛋白靶向的另外两种细胞内微环境， 宿主细胞质和包涵体腔。 这些努力有望获得宿主和衣原体蛋白质的详细蛋白质组图谱， 特别是招募到早期和晚期的包涵体膜。这项技术捕捉 原位相互作用蛋白质代表了对先前蛋白质组学研究的重大改进。我们预计 该项目产生的丰富数据将产生对细胞途径和分子因素的新见解 在感染过程中被衣原体攻击，并将提供数十年的技术和生物资源。 今后的研究重点是确定该项目确定的目标的分子机制。 最后，我们预计这项技术可以广泛地应用于其他细菌系统， 研究其分泌的效应物和宿主靶标。

PROJECT SUMMARY / ABSTRACT The major human pathogen Chlamydia trachomatis is an obligate intracellular bacterium that spends its entire replicative growth cycle within cellular vacuoles called inclusions. The chlamydial inclusion plays critical roles in establishing and maintaining a favorable intracellular niche for Chlamydia, and through the outer inclusion membrane of this vacuole, chlamydiae secrete numerous effector molecules into the host cell. The identity, function, and host interaction targets of the majority of these secreted proteins are not known, in large part due to historical challenges associated with genetic manipulation of Chlamydia. Recent breakthroughs in Chlamydia genetics have removed some of these major obstacles. Capitalizing on these advances, we have developed a genetic–proteomic system for C. trachomatis that selectively labels proteins in close proximity to the inclusion membrane, enabling the isolation and identification of these proteins by mass spectrometry. This system allows highly specific interactive mapping of the inclusion membrane proteome, even early in infection, and therefore makes possible the discovery of the bacterial and host proteins that comprise this pathogenic compartment in situ. The primary goals of this proposal are to: (i) determine the Chlamydia and host proteins that are dynamically recruited to the inclusion membrane at three stages of vacuolar growth, and (ii) extend the development of this technique for the identification of host molecular targets at two additional intracellular microenvironments targeted by chlamydial secreted proteins— the host cytosol and the inclusion lumen. These efforts are expected to derive a detailed proteomic map of host and Chlamydia proteins that are specifically recruited to early and late-stage inclusion membranes. The ability of this technique to capture interacting proteins in situ represents a major improvement over previous proteomic studies. We expect that the rich data generated from this project will yield novel insight into the cellular pathways and molecular factors targeted by Chlamydia during infection, and will provide the technical and biological resources for decades of future research focused on characterizing the molecular mechanisms of targets identified by this project. Finally, we anticipate that this technology can be broadly translated to other bacterial systems for analogous investigations of their secreted effectors and host targets.