Proteomic Approach for Protein Expression Profiling of Human Promyelocytic Cells in Response to Infection with Granulocyte- and Monocyte-Tropistic Obligatory Intracellular Microorganisms

嗜吞噬细胞无形体菌和沙菲埃立克体是细胞内的一种专性细菌，是新出现的传染病（人粒细胞无形体病和人单核细胞埃立克体病）的病原体，它们分别感染嗜粒细胞和嗜单核/巨噬细胞的专业吞噬细胞，在许多实验室中，这两种细菌都与人早幼粒细胞细胞系HL-60保持一致。蛋白质组学方法是一种强大的技术，可以分析哺乳动物细胞系和组织、植物或细菌的几种细胞事件中的全局蛋白质表达谱。在这项研究中，我们利用最近引入的DIGE（荧光二维差异凝胶电泳）系统（GE Healthcare, Piscatawy， NJ），应用于表征宿主细胞对嗜光性芽胞杆菌和沙菲芽胞杆菌感染反应中的蛋白质表达谱，该系统使我们能够在单个凝胶中检测感染和未感染细胞之间的蛋白质表达差异。在感染后第1、2和3天的时间过程中监测蛋白质组学变化，该方法在感染后第3天发现感染和未感染细胞之间共有286个蛋白点，其丰度变化。利用MALDI TOF/TOF串联质谱技术，我们成功鉴定了HL-60细胞对嗜吞噬细胞芽胞杆菌和chafeensis感染反应的154个位点的蛋白种类。这些蛋白质种类与细胞骨架、信号转导、翻译、应激反应等有关。MetaCore Pathway分析（GeneGo Inc., St. Joseph， MI）表明，宿主细胞中与嗜吞噬细胞芽胞杆菌和沙费芽胞杆菌感染相关的候选蛋白，可能是这些细菌通过蛋白-蛋白相互作用重组宿主细胞骨架，从而获得细胞内生存的生态位。

Anaplama phagocytophilum and Ehrlichia chaffeensis are obligatory intracellular bacteria, agents of emerging infectious diseases (human granulocytic anaplasmosis and human monocytic ehrlichiosis), which infect professional phagocytes with tropism for granulocytes and monocytes/macrophages, respectively, and both are maintained with human promyelocytic cell lines HL-60 in many laboratories. Proteomic approach is a powerful technique to analyze a global protein expression profile in several cellular events of mammalian cell lines and tissues, plants, or bacteria. In this study, we applied to characterize the protein expression profile in host cell response to infection with A. phgocytophiluma and E. chaffeensis by using the most recently introduced DIGE (fluorescence two-dimensional difference gel electrophoresis) system (GE Healthcare, Piscatawy, NJ) which allowed us to detect differences in protein expression between infected and uninfected cells in a single gel. Proteomic changes were monitored in a time course on day 1, 2, and 3 postinfection and this approach revealed a total of 286 protein spots with abundance changes between infected and uninfected cells on day 3 postinfection. Using MALDI TOF/TOF tandem mass spectrometry, we successfully identified the protein species of 154 spots derived from HL-60 cells in response to infections with A. phagocytophilum and E. chafeensis. These protein species are associated with cytoskeleton, signal transduction, translation, stress response and more. MetaCore Pathway analysis (GeneGo Inc., St. Joseph, MI) suggested the candidate proteins of host cells associated with A. phagocytophilum and E. chaffeensis infections, probably these bacteria may reorganize host cell cytoskeleton through the protein-protein interaction to acquire their niches for intracellular survival.