The roles of ceramide and its derivatives in A. phagocytophilum pathogenesis

嗜吞噬细胞无浆体（Anaplasma phagocytophilum，Ap）是一种专性细胞内细菌，其引起出现并潜在地 致命感染，人粒细胞无形体病（HGA）。促进Ap的微生物-宿主相互作用 细胞内增殖和扩散仍然没有得到很好的表征。我们发现了一个新的角色， 宿主生物活性鞘脂，神经酰胺-1-磷酸（C1 P），作为高尔基体片段化的调节剂。具体地说， C1 P激活PKC β/Cdc 42/JNK信号传导轴，使GRASP 55磷酸化（高尔基体重组堆积 蛋白质55 kDa），以使trans-Golgi网络（TGN）不稳定并放大TGN顺行运输。我们确定 Ap感染上调C1 P合成，并诱导高尔基体断裂，这是C1 P信号依赖性的。 方式多泡体（MVB）是接受高尔基体运输的内体分选站。MVB限幅 膜内陷和断裂将货物递送到管腔内囊泡（ILV）中的MVB中。ILV膜 富含胆固醇、鞘脂和促进胆固醇释放的鞘脂代谢酶 从ILV。MVB与质膜融合释放ILV作为外泌体，其诱导一系列的免疫反应。 受体细胞的反应，包括信号传导、免疫和炎症。外泌体的重要性- 在传染病中介导的细胞间通讯才刚刚开始受到重视。我们发现 AP存在于病原体修饰的MVB中，其接收富含鞘磷脂的TGN货物作为ILV。细菌 利用MVB脂质代谢机制寄生于鞘脂和胆固醇，这是其生长所必需的 以及从复制型向感染型的转变与MVB外泌体释放一致，Ap空泡 以Rab 27 a依赖的方式与质膜融合，将感染性子代分散到幼稚细胞中。 我们还发现，Ap改变外泌体含量。在这次竞争性的更新中，我们将质疑我们的假设 Ap诱导C1 P使高尔基体不稳定，并促进富含SM的囊泡的TGN至ApV运输， ap寄生于宿主脂质体中进行增殖和播散。我们还将检验我们的假设，即Ap调节 ILV的蛋白质组和脂质组含量，当作为外泌体与后代细菌一起释放时， 协同有益于幼稚细胞感染和/或有助于与HGA相关的免疫病理学。 完成本文的目的将产生细胞内细菌发病机制的最精细模型之一， 进一步阐明了我们新发现的C1 P作为高尔基体稳定性调节因子和微生物靶点的作用， 宿主调节，并进一步了解外泌体如何有助于致病过程。

Anaplasma phagocytophilum (Ap) is an obligate intracellular bacterium that causes the emerging and potentially fatal infection, human granulocytic anaplasmosis (HGA). The microbial-host interactions that facilitate Ap intracellular proliferation and dissemination have remained poorly characterized. We uncovered a novel role for the host bioactive sphingolipid, ceramide-1-phosphate (C1P), as a regulator of Golgi fragmentation. Specifically, C1P activates a PKC/Cdc42/JNK signaling axis that phosphorylates GRASP55 (Golgi reassembly stacking protein 55 kDa) to destabilize the trans-Golgi network (TGN) and amplify TGN anterograde traffic. We determined that Ap infection upregulates C1P synthesis and induces Golgi fragmentation in a C1P signaling-dependent manner. Multivesicular bodies (MVBs) are endosomal sorting stations that receive Golgi traffic. MVB limiting membrane invagination and scission deliver cargo into the MVB in intraluminal vesicles (ILVs). ILV membranes are enriched in cholesterol, sphingolipids, and sphingolipid metabolic enzymes that promote cholesterol release from ILVs. MVB fusion with the plasma membrane releases ILVs as exosomes, which induce a battery of responses in recipient cells including signaling, immunity, and inflammation. The importance of exosome- mediated cell-to-cell communication in infectious disease is just beginning to be appreciated. We discovered that Ap resides in a pathogen-modified MVB that receives sphingomyelin-rich TGN cargo as ILVs. The bacterium coopts MVB lipid metabolic machinery to parasitize sphingolipids and cholesterol, which is essential for its growth and conversion from its replicative to infectious form. Consistent with MVB exosome release, the Ap vacuole fuses with the plasma membrane in a Rab27a-dependent manner to disperse infectious progeny to naïve cells. We also found that Ap alters exosome content. In this competitive renewal, we will interrogate our hypothesis that Ap induces C1P to destabilize the Golgi and promote TGN-to-ApV trafficking of SM-rich vesicles that enables Ap to parasitize host lipids for proliferation and dissemination. We will also test our hypothesis that Ap modulates the proteomic and lipidomic content of ILVs that, when released as exosomes together with progeny bacteria, synergistically benefit infection of naïve cells and/or contributes to immunopathology associated with HGA. Completing the Aims herein will yield one of the most refined models for intracellular bacterial pathogenesis, further illuminate our newly discovered roles of C1P as both a regulator of Golgi stability and microbial target for host modulation, and further understanding of how exosomes contribute to pathogenic processes.