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

无浆体吞噬细胞性肺炎(AP)是一种专性胞内细菌，它能引起新出现的和潜在的 致命感染，人类粒细胞无形体病(HGA)。促进AP的微生物-宿主相互作用 细胞内增殖和扩散的特征仍然很差。我们发现了一个新的角色 宿主具有生物活性的鞘磷脂，神经酰胺-1-磷酸(C1P)，作为高尔基体碎裂的调节因子。具体来说， C1P激活蛋白激酶C/CDC42/JNK信号轴，使GRASP55(高尔基体重组堆积)磷酸化 蛋白质55 kDa)，以破坏跨高尔基网络(TGN)的稳定，并放大TGN顺行流量。我们决定 AP感染上调C1P合成并诱导C1P信号依赖的高尔基体碎裂 举止。多泡小体(MVB)是接收高尔基体运输的内体分选站。MVB限制 膜凹陷和断裂以腔内小泡(ILV)的形式将货物运送到MVB中。ILV膜 富含促进胆固醇释放的胆固醇、鞘脂和鞘脂代谢酶 来自ILV。MVB与质膜的融合将ILV释放为外体，从而诱导一系列 受体细胞的反应包括信号、免疫和炎症。外显体的重要性-- 感染性疾病中介导的细胞间通讯才刚刚开始受到重视。我们发现 AP驻留在病原体修饰的MVB中，该MVB接收富含鞘磷脂的TGN货物作为ILV。细菌 Coopts MVB脂代谢机制寄生鞘脂和胆固醇，这是其生长所必需的 以及从复制型转变为感染型。与MVB外切体释放一致，AP空泡 以Rab27a依赖的方式与质膜融合，将感染性后代分散到幼稚细胞。 我们还发现，AP会改变外显体的内容。在这场竞争性更新中，我们将质疑我们的假设 AP诱导C1P破坏高尔基体的稳定，促进TGN到APV的富含SM的囊泡的运输，从而使 AP寄生宿主脂类以进行增殖和传播。我们还将测试我们的假设，即AP调节 ILV的蛋白质组和脂体组内容，当与后代细菌一起作为外体释放时， 协同作用有利于幼稚细胞的感染和/或有助于与HGA相关的免疫病理。 完成这里的目标将产生细胞内细菌致病最精细的模型之一， 进一步阐明我们新发现的C1P作为高尔基体稳定性调节剂和微生物靶标的作用 寄主调节，以及进一步了解外切体如何在致病过程中起作用。

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.