Ubiquitylation and Rickettsial Colonization of a Tick Vector

 描述（由申请人提供）：结合功能测定的比较基因组学分析揭示了进化上保守的信号传导途径，并为理解昆虫免疫提供了重要见解。然而，将这种方法外推到非昆虫节肢动物，例如蜱类肩突硬蜱，有时会构成问题，因为缺乏可区分的蛋白质同源物和由于特定基因组的低序列覆盖率或不完整性而导致的基因的不正确注释。例如，我们观察到蜱I的免疫缺陷（IMD）信号通路。尽管在其基因组上缺乏衔接分子imd和基因fadd，但肩胛骨对于防御立克次体病原体嗜吞噬细胞无形体是关键的。此外，我们了解到E3泛素连接酶X连锁凋亡抑制蛋白（XIAP）对于激活IMD通路对抗A。嗜吞噬细胞I.肩胛肌蜱虫总的来说，这些发现导致了一个中心假设，即XIAP调节非经典蜱IMD途径以响应立克次体因子A。嗜吞噬细胞菌因此，在本提案的目标#1中，我们将表征XIAP和E2缀合酶Bendless之间的功能性相互作用。通过结构对接和质谱分析，我们发现这两种蛋白质之间存在直接的相互作用。在目标#2中，我们将使用细胞培养物中氨基酸的稳定同位素标记（SILAC）和双甘氨酸残基亲和力分析结合串联质谱法鉴定蜱病原体定殖期间的XIAP底物。在目标#3中，我们将在A期间验证IMD网络。嗜吞噬细胞I.肩胛蜱体内。我们将确定病原体进入蜱后的免疫反应，并评估病原体感染期间IMD信号通路的影响。这项研究的结果将是双重的：（1）它将提供与蜱-病原体相互作用相关的基础知识;（2）它将揭示节肢动物中一种新的免疫途径，对媒介生物学界具有广泛的影响。

 DESCRIPTION (provided by applicant): Comparative genomics analysis coupled to functional assays uncovered evolutionarily conserved signaling pathways and provided important insights towards the understanding of insect immunity. However, extrapolating this approach to non-insect arthropods, such as the tick Ixodes scapularis, sometimes constitutes a problem because of the lack of distinguishable protein homologues and incorrect annotation of genes due to low sequence coverage or incompleteness of a particular genome. For instance, we observed that the immune deficiency (IMD) signaling pathway of the tick I. scapularis is critical for defense against the rickettsial agent Anaplasma phagocytophilum despite the absence of the adaptor molecule imd and the gene fadd on its genome. Moreover, we learned that the E3 ubiquitin ligase x-linked inhibitor of apoptosis protein (XIAP) is critical for activating the IMD pathway against A. phagocytophilum infection of I. scapularis ticks. Collectively, these findings led to ou central hypothesis stating that XIAP regulates the non-canonical tick IMD pathway in response to the rickettsial agent A. phagocytophilum. Accordingly, in Aim #1 of this proposal, we will characterize the functional interaction between XIAP and the E2-conjugating enzyme Bendless. We surmise a direct interaction between these two proteins based on structural docking and mass spectrometry analysis. In Aim #2, we will identify XIAP substrates during pathogen colonization of ticks using stable isotope labeling with amino acids in cell culture (SILAC) and diglycine remnant affinity profiling combined with tandem mass spectrometry. In Aim #3, we will validate the IMD network during A. phagocytophilum infection of I. scapularis ticks in vivo. We will ascertain the immune response following pathogen entry into the tick and evaluate the effect of the IMD signaling pathway during pathogen infection. The outcome of this research will be two-fold: (1) it will provide fundamental knowledge related to tick-pathogen interactions; and (2) it will uncover a novel immune pathway in arthropods with broad implications for the vector biology community.