T4SS effectors and tick tropism in Anaplasma phagocytolium

项目摘要 壁虱传播的疾病正在增加，并且是几乎所有媒介传播疾病的罪魁祸首。 在美国。病媒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： 节肢动物媒介和哺乳动物宿主。为了在真核细胞中生存，立克次体病原体 无浆体吞噬细胞抑制吞噬溶酶体成熟，抑制细胞凋亡，调节宿主基因 表达，重定向跨高尔基体贩卖，并改变自噬机制的用途，以建立复制空泡。 吞噬细胞必须做到这一点，同时躲避哺乳动物独特的先天免疫防御。 和节肢动物宿主细胞。立克次体菌目的所有病原体都利用专门的IV型分泌系统 (T4SS)将效应分子运送到宿主细胞胞浆中，以介导宿主病原体的相互作用。然而， 由于这些病原体的专性性质，对分泌效应物的鉴定一直受到限制。更少的是 已知效应器如何促进扁虱细胞中立克次体的生长，因为扁虱载体仍未得到充分研究 这些病原体的生态位。为了克服这个问题，我们的团队开发了一个T4SS效应器预测程序 T4SS效应器的最佳特性预测器(OPT4e)。当应用于吞噬细胞菌时，OPT4e被鉴定为 48个推定的T4SS效应器。转录组学发现，这些预测的效应器基因中有15个是特定的 在扁虱或哺乳动物细胞内的生长过程中表达。我们已经证明了其中一只扁虱- 特定的效应候选者Aph1383由嗜肺军团菌以T4特异的方式转运 T4SS。Aph1383也属于由基因簇ap1380-1386编码的六种蛋白的平行家族。 在扁虱细胞中吞噬细胞的生长过程中，这一整个簇的表达水平比在 哺乳动物细胞感染。我们假设这一家族的Aph1383鹦鹉都是T4SS效应器 目标性宿主细胞过程对扁虱细胞内的吞噬阿糖胞菌的生长特别重要。在这项研究中，我们 将首次使用在ap1380-1386基因簇中的转座子插入突变体来测试 这些基因在扁虱细胞内吞噬嗜血杆菌的生长过程中。接下来，我们将评估T4SS易位 所有Aph1383的同源序列，并确定分泌所需的氨基酸序列。最后，我们将确定 Aph1383的亚细胞定位和分子靶点。描述这些分子的特征 扁虱细胞内吞噬弧菌的相互作用将为靶向载体的开发打开大门 减少病原体传播性的干预措施。

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.