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

专性胞内细菌是全世界感染性疾病的主要原因，其发病率和 严肃性。它们寄生宿主细胞代谢物以求生存的破译机制将会取得进展 了解微生物的发病机制，并可能导致开发新的治疗方法 它们引起的感染。无形体吞噬细胞性肺炎(AP)是一种专性胞内细菌 导致潜在的致命人畜共患病，人类粒细胞无浆症。我们发现美联社 将富含鞘磷脂的囊泡从跨高尔基网络(TGN)劫持到其液泡中。 以依赖的方式推动从非传染性形式向传染性形式的转变。AP感染性子代 富含宿主鞘磷脂神经酰胺，神经酰胺由酸性鞘磷脂酶(ASMase)产生- 神经鞘蛋白的介导性水解性。值得注意的是，宿主ASMase也被路由到AP液泡(APV)。 敲除或抑制Rab10或ASMase可阻止AP感染循环，AP不能有效地 感染ASMase-/-小鼠。由于AP寄生TGN囊泡，因此TGN顺行流量的增加将 有益于感染。事实上，我们发现AP通过上调宿主细胞来诱导这种现象 生产具有生物活性的鞘磷脂，Cerk衍生的神经酰胺-1-磷酸(C1P)。引人注目的是， C1P通过与UVRAG的相互作用诱导高尔基体失稳和顺行交通诱导。 我们假设AP在高尔基体诱导C1P的形成，高尔基体招募UVRAG来诱导 APV截取的Rab10阳性、富含鞘磷脂的囊泡的顺行运输。我们进一步 假设在APV，被劫持的ASMase将神经鞘磷脂转化为神经酰胺，从而推动感染性 后代生产。目标1将确定为什么Rab10对AP毒力至关重要。目标2将定义 ASMase和神经酰胺在AP病理生物学中的作用目标3将确定C1P和UVRAG在 利用新的转基因小鼠模型劫持TGN流量和体内AP感染。C1P的角色是 最近出现了包括癌症和炎症在内的多种细胞过程的强有力的调节器。 在这里，我们将第一次阐明C1P在传染病中也发挥着关键作用。这个 我们的研究结果将确定神经酰胺的新的和以前未曾推测的机制 胞内细菌的寄生。如果我们的假设得到证实，ASMase和CERK将成为目标 开发针对这些类型病原体的新一代疗法。总的来说，这项工作将 产生广泛而强大的影响。

Obligate intracellular bacteria are major causes of infectious disease worldwide in terms of incidence and severity. Deciphering mechanisms by which they parasitize host cell metabolites to survive will advance understanding of microbial pathogenesis and may lead to development of novel therapeutics for the infections that they cause. Anaplasma phagocytophilum (Ap) is an obligate intracellular bacterium that causes the potentially deadly zoonosis, human granulocytic anaplasmosis. We discovered that Ap hijacks sphingomyelin-rich vesicles from the trans-Golgi network (TGN) to its vacuole in a Rab10- dependent manner to drive conversion from its non-infectious to infectious form. Ap infectious progeny are enriched in the host sphingolipid, ceramide, which is produced by acid sphingomyelinase (ASMase)- mediated hydrolysis of sphingomyelin. Notably, host ASMase is also routed to the Ap vacuole (ApV). Knocking down or inhibiting Rab10 or ASMase arrests the Ap infection cycle, and Ap cannot productively infect ASMase-/- mice. Since Ap parasitizes TGN vesicles, an increase in TGN anterograde traffic would benefit infection. Indeed, we discovered that Ap induces this very phenomenon by upregulating host cell production of the bioactive sphingolipid, CERK-derived ceramide-1-phosphate (C1P). Conspicuously, C1P induces Golgi destabilization and anterograde traffic induction through its interaction with UVRAG. We hypothesize that Ap induces C1P formation at the Golgi, which recruits UVRAG to induce anterograde trafficking of Rab10-positive, sphingomyelin-rich vesicles that the ApV intercepts. We further posit that, at the ApV, hijacked ASMase converts sphingomyelin to ceramide, which drives infectious progeny production. Aim 1 will determine why Rab10 is critical for Ap virulence. Aim 2 will define the roles of ASMase and ceramide in Ap pathobiology. Aim 3 will determine the roles of C1P and UVRAG in hijacking TGN traffic and Ap infection in vivo using novel transgenic mouse models. C1P’s role as a potent regulator of diverse cellular processes including cancer and inflammation has recently emerged. Here, we stand to illuminate for the first time that C1P also plays a critical role in infectious disease. The culmination of our studies will define novel and previously unsurmised mechanisms for ceramide parasitism by intracellular bacteria. If our hypotheses are validated, ASMase and CERK become targets for developing new generations of therapeutics against these types of pathogens. Overall, this work will have a broad and powerful impact.