High throughput proteomics to dissect Chlamydia-host cell interactions

描述（由申请方提供）：衣原体属是人类疾病的重要原因，目前尚无疫苗。标准的遗传学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。最近的变革性研究表明，尽管其基因组大小减少，约1000个基因，衣原体分泌远远超过100个效应蛋白进入宿主细胞。这些效应子的功能是选择性地募集细胞器，获得营养，操纵宿主细胞运输途径，并逃避宿主免疫系统的检测。系统地表征被入侵并在哺乳动物宿主细胞内复制的病原体物理劫持的宿主蛋白质是理解其生物学的关键。以前在包括芽殖酵母和大肠杆菌在内的简单系统中的研究已经证明，这种奋进在揭示新的生物学见解方面是非常强大的。我们提出了两个具体的目标，其中我们将使用最先进的高通量质谱与新开发的生物信息学算法相结合，以（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，以及（ii）全面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的变化，泛素化和糖基化事件。目标1.我们将在全球范围内确定分泌型C。沙眼衣原体蛋白及其宿主细胞靶蛋白通过亲和纯化/质谱（AP/MS），以建立全面的C.沙眼-宿主蛋白质-蛋白质相互作用（PPI）图。目标2.我们将使用一种无偏倚的方法来全面鉴定宿主翻译后修饰的变化，包括宿主蛋白磷酸化、泛素化和糖基化，以响应C。沙眼感染使用这些创新的方法解开这些复杂的事件将产生重要的线索，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。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.