The ejectosome: Deciphering its proteome

喷射体：破译其蛋白质组

• 批准号: 445702567
• 负责人: Dr. Monica Hagedorn
• 金额: --
• 依托单位: HMU Research, Development und Innovation gGmbH (HMU RDI)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/445702567?language=en
• 关键词: ejectosome; Deciphering; its; proteome

Cell-to-cell transmission of pathogenic organisms is equally important to maintain and spread an efficient infection as uptake and replication. However, little is known about the underlying molecular mechanisms, which allow pathogens to spread efficiently from cell to cell.In the Dictyostelium- Mycobacterium marinum infection system the ejectosome, an actin-rich structure, represents the major mechanism of efficient cell-to-cell spreading. Ejection allows bacteria to exit their host cell directly from the cytosol through the plasma membrane in a non-lytic fashion. Little is known about the underlying molecular mechanism and about involved factors of the pathogen as well as host.In the proposed project we plan to use two major approaches to decipher the protein composition of the ejectosome structure. First, we plan to express host and pathogen proteins, which we know are present in the ejectosome, as fusions to APEX2. Proteins, that come in close contact with these "bait-proteins" will be biotinylated and thus can be isolated and subsequently identified. In another approach we plan to make use of a Dictyostelium strain, that overexpresses an actin-binding domain which not only results in a higher number of ejectosomes but also more stable ejectosome structures. By optimizing the lysis conditions and subsequent enrichment steps we plan to purify ejectosomes, the protein composition of which then can be deciphered applying mass spectrometric analyses.We expect that deciphering the proteome of the ejectosome will allow us to identify host and pathogen factors, which take part in the cell-to-cell transmission of bacteria and thereby elucidate the underlying molecular mechanism of this process. Furthermore, the acquired knowledge will highlight new strategies to contain infections on the cellular level, by interfering with egress. This will represent a further and complementary approach to counteract infections.

病原生物体的细胞间传播对于维持和传播有效感染与摄取和复制同样重要。然而，人们对病原体在细胞间有效传播的潜在分子机制知之甚少。在盘基网柄菌-海分枝杆菌感染系统中，喷射体（一种富含肌动蛋白的结构）代表了细胞间有效传播的主要机制。喷射允许细菌以非裂解方式通过质膜直接从细胞质中离开宿主细胞。对于潜在的分子机制以及病原体和宿主的相关因素知之甚少。在拟议的项目中，我们计划使用两种主要方法来破译喷射体结构的蛋白质组成。首先，我们计划表达宿主和病原体蛋白，我们知道这些蛋白存在于喷射体中，作为 APEX2 的融合体。与这些“诱饵蛋白”密切接触的蛋白质将被生物素化，因此可以被分离并随后进行鉴定。在另一种方法中，我们计划利用盘基网柄菌属菌株，该菌株过度表达肌动蛋白结合域，这不仅会产生更多数量的喷射体，而且会产生更稳定的喷射体结构。通过优化裂解条件和随后的富集步骤，我们计划纯化喷射体，然后可以应用质谱分析破译其蛋白质组成。我们期望破译喷射体的蛋白质组将使我们能够识别参与细菌细胞间传播的宿主和病原体因子，从而阐明该过程的潜在分子机制。此外，所获得的知识将突出通过干扰出口来控制细胞水平感染的新策略。这将是对抗感染的进一步补充方法。