Ubiquitylation and Rickettsial Colonization of a Tick Vector

 描述(申请人提供)：与功能分析相结合的比较基因组分析揭示了进化上保守的信号通路，并为了解昆虫免疫提供了重要的见解。然而，将这种方法外推到非昆虫节肢动物，如扁虱，有时会构成一个问题，因为缺乏可区分的蛋白质同源物，以及由于序列覆盖率低或特定基因组的不完整而对基因的错误注释。例如，我们观察到，尽管在其基因组上缺乏适配分子IMD和FADD基因，但扁虱的免疫缺陷(IMD)信号通路对于防御立克次体病原体吞噬无形体是至关重要的。此外，我们还了解到，E3泛素连接酶X连锁的凋亡抑制蛋白(XIAP)对于激活IMD途径以对抗伊蚊的吞噬细胞感染至关重要。总而言之，这些发现导致了我们的中心假说，即XIAP调节非典型性扁虱IMD途径，以响应立克次体病原体A吞噬细胞。因此，在本提案的目标#1中，我们将表征XIAP和E2结合酶Bend之间的功能相互作用。基于结构对接和质谱分析，我们推测这两种蛋白质之间存在直接相互作用。在目标2中，我们将使用细胞培养中氨基酸的稳定同位素标记(SILAC)和二甘氨酸残基亲和图谱结合串联质谱仪来鉴定硬蜱病原体定植过程中的XIAP底物。在目标3中，我们将在体内验证吞噬伊蚊过程中的IMD网络。我们将确定病原体进入硬蜱后的免疫反应，并评估IMD信号通路在病原体感染过程中的作用。这项研究的结果将有两个方面：(1)它将提供与扁虱-病原体相互作用相关的基本知识；(2)它将揭示节肢动物中一种新的免疫途径，对媒介生物界具有广泛的意义。

 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.