T4SS effectors and tick tropism in Anaplasma phagocytolium

项目摘要 蜱传播的疾病正在增加，几乎所有的病媒传播疾病都是蜱传播的 在美国.媒介传播的病原体面临着适应两种截然不同的宿主环境的双重挑战： 节肢动物载体和哺乳动物宿主。为了在真核细胞中生存，立克次体病原体 嗜吞噬细胞无形体阻断吞噬溶酶体成熟，抑制凋亡，调节宿主基因 表达，重定向trans-Golgi运输，并重新利用自噬机制来构建复制泡。 A.嗜吞噬细胞菌必须做到这一点，同时逃避哺乳动物独特的先天免疫防御， 和节肢动物宿主细胞。立克次体目中的所有病原体都利用专门的IV型分泌系统 T4 SS）将效应分子递送到宿主细胞胞质溶胶中以介导宿主病原体相互作用。但是，在这方面， 分泌的效应物的鉴定受到这些病原体的专性性质的限制。更不是 已知效应子如何促进蜱细胞中立克次体的生长，因为蜱载体仍然是一个研究不足的领域。 这些病原体的生态位。为了克服这一点，我们的小组已经开发了一个T4 SS效应预测程序， T4 SS效应器的最佳特征预测器（OPT 4 e）。当应用于A.嗜吞噬细胞菌，OPT 4 e鉴定 48个T4 SS效应子转录组学发现，这些预测的效应基因中有15个是特异性的。 在蜱或哺乳动物细胞内生长期间表达。我们已经证明了其中一种蜱虫- 特异性效应子候选物Aph 1383以T4特异性方式被嗜肺军团菌易位 T4SS aph 1383也属于由基因簇aph 1380 -1386编码的六种蛋白质的旁系同源家族。 这整个簇在A.蜱细胞中嗜吞噬细胞的生长比 哺乳动物细胞感染。我们假设这个Aph 1383旁系同源家族都是T4 SS效应子， 靶向宿主细胞过程对A.蜱细胞内嗜吞噬细胞生长。本研究 将首先使用aph 1380 -1386基因簇中的转座子插入突变体来测试 这些基因在A.蜱细胞内嗜吞噬细胞生长。接下来，我们将评估T4 SS易位， 所有Aph 1383旁系同源物，并鉴定分泌所必需的氨基酸序列。最后，我们将确定 Aph 1383在蜱细胞内的亚细胞定位和分子靶点。表征这些分子 A的相互作用。蜱细胞内的嗜吞噬细胞菌将为靶向载体的开发打开大门 干预措施，以减少病原体的传播。

Project Summary Tick-borne diseases are on the increase, and are responsible for nearly all of the vector-transmitted disease in the US. Vector-borne pathogens face the dual challenge of adaptation to two very different host environments: the arthropod vector and the mammalian host. To survive within eukaryotic cells, the rickettsial pathogen Anaplasma phagocytophilum blocks phago-lysosome maturation, inhibits apoptosis, modulates host gene expression, redirects trans-Golgi trafficking, and repurposes autophagic machinery to build a replication vacuole. A. phagocytophilum must accomplish this, all while evading the unique innate immune defenses of mammalian and arthropod host cells. All pathogens in the order Rickettsiales utilize a specialized Type IV Secretion System (T4SS) to deliver effector molecules into the host cell cytosol to mediate host pathogen interactions. However, identification of the secreted effectors has been limited by the obligate nature of these pathogens. Even less is known about how effectors contribute to rickettsial growth in tick cells, as the tick vector remains an understudied niche of these pathogens. To overcome this, our group has developed a T4SS effector prediction program Optimal-features Predictor for T4SS Effectors (OPT4e). When applied to A. phagocytophilum, OPT4e identified 48 putative T4SS effectors. Transcriptomics finds that 15 of these predicted effector genes are specifically expressed during growth within either tick or mammalian cells. We have demonstrated that one of these tick- specific effector candidates, Aph1383, is translocated in a T4 specific manner by the Legionella pneumophila T4SS. Aph1383 also belongs to a paralogous family of six proteins encoded by the gene cluster aph1380-1386. This entire cluster is expressed 2.5-fold more highly during A. phagocytophilum growth in tick cells than during mammalian cell infections. We hypothesize that this family of Aph1383 paralogs are all T4SS effectors which target host cell processes specifically important for A. phagocytophilum growth within tick cells. In this study, we will first use transposon insertion mutants in the aph1380-1386 gene cluster to test the fitness contribution of these genes during A. phagocytophilum growth within tick cells. Next, we will evaluate the T4SS translocation of all Aph1383 paralogs and identify the amino acid sequences necessary for secretion. Finally, we will identify the subcellular localization and molecular targets of Aph1383 within tick cells. Characterizing these molecular interactions of A. phagocytophilum within the tick cell will open the door to development of vector targeted interventions to reduce transmissibility of the pathogen.