PROTEOMIC DIS. OF CHLAMYDIA INCLUSION-HOST INTERACTIONS

描述：沙眼衣原体是一种专性细胞内病原体，是美国性传播疾病的主要原因，也是第三世界国家可预防失明的主要原因。这种寄生虫进入非专业吞噬上皮细胞并在真核细胞质的细胞内环境中存活的能力是其发病机制的关键。据推测，衣原体与其宿主之间的成功相互作用依赖于早期从头合成的衣原体蛋白，这些蛋白在包涵体膜的结构和功能重塑中发挥作用，从而使宿主的降解途径得以规避。然而，这些蛋白质的特性和相互作用的分子机制要么未知，要么知之甚少。这项建议旨在鉴定参与包涵体膜重塑的衣原体蛋白，并表征它们与宿主细胞蛋白的相互作用。具体目标1建议用稳定的同位素进行代谢标记，然后用多维蛋白质鉴定技术(MudPIT)进行蛋白质组学分析，从而鉴定这些衣原体蛋白。特异性目的2利用亚细胞分离和消减蛋白质组学相结合的方法来筛选定位于包涵膜细胞质表面的衣原体蛋白。最后，特异性目标3将聚焦于从特异性目标2中筛选出的衣原体蛋白，并产生重组融合蛋白，以在体外鉴定相互作用的宿主蛋白。

Description: Chlamydia trachomatis is an obligate intracellular pathogen that is the leading cause of sexually transmitted diseases in the United States and the major cause of preventable blindness in third world countries. The ability of this parasite to enter non-professional phagocytic epithelial cells and survive within the intracellular environment of the eukaryotic cytoplasm is the key to its pathogenesis. It is hypothesized that successful interaction between chlamydia and its host is dependent on early de novo chlamydial protein synthesis and that these proteins play a role in the structural and functional remodeling of the inclusion membrane thereby enabling the evasion of host degradation pathways. However, the identities of these proteins and the molecular mechanisms of interaction are either unknown or poorly understood. This proposal is designed to identify chlamydial proteins involved in the remodeling of the inclusion membrane and to characterize their interactions with host cell proteins. Specific Aim 1 proposes to identify these chlamydial proteins by metabolic labeling with stable isotopes followed by proteomic analysis using Multi-dimensional Protein Identification Technology (MudPIT). Specific Aim 2 utilizes subcellular fractionation coupled with subtractive proteomics to select for chlamydial proteins localized to the cytoplasmic surface of the inclusion membrane. Finally, Specific Aim 3 will focus on the selected chlamydial proteins from Specific Aim 2 and generate recombinant fusion proteins to identify interacting host proteins in vitro.