High throughput proteomics to dissect Chlamydia-host cell interactions

描述（由申请人提供）：衣原体是人类疾病的重要病因，目前尚无疫苗。标准的遗传方法不可能用于这种专性细胞内寄生虫，阻碍了我们解码其分子发病机制的能力。最近的转化研究表明，尽管衣原体的基因组大小减少了，大约有1000个基因，但它向宿主细胞分泌了100多种效应蛋白。这些效应物的功能是选择性地招募细胞器，获取营养物质，操纵宿主细胞运输途径，并逃避宿主免疫系统的检测。系统地描述被病原体入侵并在哺乳动物宿主细胞内复制的宿主蛋白质是了解其生物学的关键。以前对更简单的系统的研究，包括出芽酵母和大肠杆菌，已经证明了这种努力在发现新的生物学见解方面是非常强大的。我们提出了两个具体目标，我们将使用最先进的高通量质谱技术结合新开发的生物信息学算法来(i)全面鉴定与分泌的衣原体效应物相互作用的宿主蛋白质；（ii）全球鉴定响应衣原体感染的宿主翻译后修饰，包括宿主蛋白质组的总体变化以及宿主蛋白质磷酸化、泛素化的变化。还有糖基化事件。目的1。我们将通过亲和纯化/质谱（AP/MS）技术在全球范围内鉴定沙眼衣原体分泌蛋白与其宿主细胞靶蛋白之间的相互作用，建立全面的沙眼衣原体-宿主蛋白-蛋白相互作用（PPI）图谱。目标2。我们将使用一种无偏见的方法来全球识别宿主对沙眼衣原体感染的翻译后修饰的变化，包括宿主蛋白磷酸化、泛素化和糖基化。利用这些创新的方法解开这些复杂的事件将为衣原体呼吸道感染的发病机制、衣原体感染在动脉粥样硬化性心脏病中的作用以及衣原体感染在肺癌中的作用提供重要的线索。这些研究可能为诊断、治疗和疫苗提供新的靶点。此外，这些研究将为真核细胞生物学的基本过程提供新的见解，其影响范围从发育生物学到癌症生物学。

DESCRIPTION (provided by applicant): Chlamydia species are an important cause of human disease for which no vaccine exists. Standard genetic approaches are not possible to employ with this obligate intracellular parasite, hampering our ability to decode its molecular pathogenesis. Recent transformative studies have revealed that despite its reduced genome size, on the order of 1000 genes, Chlamydia secretes well over 100 effector proteins into the host cell. These effectors function to selectively recruit organelles, acquire nutrients, manipulat host cell trafficking pathways, and to evade detection from the host immune system. Systematically characterizing the host proteins that are physically hijacked by pathogens that invade and replicate within the mammalian host cell is key to understanding their biology. Previous studies in simpler systems, including budding yeast and Escherichia coli, have demonstrated that such an endeavor is incredibly powerful with respect to uncovering novel biological insights. We propose two specific aims in which we will use state-of-the art high-throughput mass spectroscopy in conjunction with newly developed bioinformatic algorithms to (i) comprehensively identify host proteins that interact with secreted chlamydial effectors and to (ii) globally identify host post-translational modifications in response to chlamydial infections, including overall changes in the host proteome as well as changes in host protein phosphorylation, ubiquitylation, and glycosylation events. Aim 1. We will globally identify interactions between secreted C. trachomatis proteins and their host cell target proteins by affinity purification/mass spectrometry (AP/MS) to develop a comprehensive C. trachomatis-host protein-protein interaction (PPI) map. Aim 2. We will use an unbiased approach to globally identify changes in host post-translational modifications, including host protein phosphorylation, ubiquitylation and glycosylation, in response to C. trachomatis infection. Unraveling these complex events using these innovative approaches will yield important clues into the pathogenesis of Chlamydia respiratory infections, the role of Chlamydia infections in atherosclerotic heart disease, and the role of Chlamydia infections in lung cancer. The studies may provide new targets for diagnostics, therapeutics, and vaccines. In addition, these studies will provide novel insights into fundamental process in eukaryotic cell biology, with implications ranging from developmental biology to cancer biology.