High throughput proteomics to dissect Chlamydia-host cell interactions

说明(申请人提供)：衣原体是人类疾病的一个重要原因，目前尚无疫苗可供预防。对于这种专性的细胞内寄生虫，标准的遗传方法是不可能使用的，这阻碍了我们破译其分子发病机制的能力。最近的变革性研究表明，尽管衣原体的基因组规模缩小了，大约有1000个基因，但它能向宿主细胞分泌100多种效应蛋白。这些效应器的功能是选择性地招募细胞器，获取营养，操纵宿主细胞的运输途径，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是了解其生物学的关键。以前在更简单的系统中的研究，包括发芽酵母和大肠杆菌，已经证明这种努力在发现新的生物学见解方面是令人难以置信的强大的。我们提出了两个具体的目标，在这两个目标中，我们将使用最先进的高通量质谱学和新开发的生物信息学算法来(I)全面地识别与分泌型衣原体效应器相互作用的宿主蛋白，以及(Ii)全局地识别宿主对衣原体感染的翻译后修饰，包括宿主蛋白质组的整体变化以及宿主蛋白磷酸化、泛素化和糖基化事件的变化。目的1.通过亲和纯化/质谱法(AP/MS)鉴定沙眼衣原体分泌蛋白与宿主细胞靶蛋白之间的相互作用，建立沙眼衣原体-宿主蛋白-蛋白相互作用(PPI)图谱。目的2.我们将使用一种无偏见的方法来全局识别宿主蛋白翻译后修饰的变化，包括宿主蛋白磷酸化、泛素化和糖基化，以响应沙眼衣原体感染。使用这些创新的方法揭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.