Host-C. trachomatis Interaction by muLc-Ms/Ms

描述（由申请人提供）：沙眼衣原体感染真核宿主细胞是由初级体（EBs）附着开始的，随后内吞进入膜结合液泡（包涵体）。早期衣原体基因产物的表达（感染后2小时内）对于包裹体向高尔基周围区域的物理运输以及与含有鞘磷脂的后高尔基囊泡的融合能力的建立都是必需的。这种与胞吐途径的物理相互作用被认为是包涵体从溶酶体途径的降解中分离出来的机制，并构成了一个支持寄生虫复制的保护位点。了解宿主细胞的分子反应以及细菌蛋白表达的时间序列对于理解宿主与细菌寄生虫之间的相互作用至关重要。传统上，这种类型的蛋白质分析是使用放射性代谢标记和比较二维凝胶技术完成的。由于灵敏度、定量和检测翻译后修饰的限制，该方法并不健壮。该项目的一个主要目的是开发使用质谱法快速分析细胞总裂解液混合物中差异表达蛋白及其翻译后修饰的方法。这些方法将用于监测衣原体进入、包涵体的建立以及随后在周围高尔基区发生的融合事件时宿主细胞的蛋白质表达和翻译后修饰的变化。此外，细菌蛋白表达将在这些时间点上进行时间分析。这些新的蛋白质组学方法将为随后的分子解剖确定宿主和寄生虫之间的功能相互作用确定关键分子。

DESCRIPTION (provided by applicant): Infection of the eukaryotic host cell by Chlamydia trachomatis is initiated by the attachment of elementary bodies (EBs) followed by endocytosis into a membrane-bound vacuole (the inclusion). Expression of early chlamydial gene products (within 2 hours after infection) is required for both the physical transport of the inclusion to the peri-Golgi region and the establishment of fusion competence with sphingomyelin-containing post-Golgi vesicles. This physical interaction with the exocytic pathway has been proposed to be the mechanism whereby the inclusion is isolated from degradation in the lysosomal pathway and constitutes a protected site supporting parasite replication. Knowledge of the molecular responses of the host cell as well as the temporal sequence of bacterial protein expression is crucial to the understanding of the interactions between host and bacterial parasite. Traditionally, this type of protein profiling has been done using radioactive metabolic labeling followed by comparative 2D gel technology. This method is not robust due to limitations in sensitivity, quantita-tion, and detection of post-translational modifications. A major aim of this project is to develop methods using mass spectrometry to rapidly profile differentially expressed proteins and their post-translational modifications in total cell lysate mixtures. These methods will be used to monitor the changes in protein expression and post-translational modifications of the host cell in response to chlamydial entry, establishment of the inclusion, and subsequent fusion events at the peri-Golgi region. Additionally, bacterial protein expression will be temporally profiled during these time points. These novel proteomic approaches will identify key molecular players for subsequent molecular dissection to determine functional interactions between host and parasite.