Mechanisms and functions of host organelle usurpation by intravacuolar Toxoplasma

摘要 脂类通过囊泡和非囊泡途径在膜之间转移。许多微生物 感染哺乳动物细胞会颠覆这些宿主细胞的脂质转运途径，从而获得脂质。 弓形虫是一种专性的细胞内寄生虫，在哺乳动物细胞的细胞质中繁殖。 一种自制的膜结合室--寄生性液泡(PV)。弓形虫的PV不是 与宿主细胞器融合。然而，我们证明了寄生虫的细胞内生存依赖于提取的脂类 来自各种哺乳动物的细胞器。例如，弓形虫从宿主体内清除胆固醇和鞘脂。 内吞细胞器和高尔基囊泡，这提出了一个令人困惑的问题，即弓形虫如何 在没有融合的情况下获取这些细胞器的脂质含量。为了解决这个问题，我们的第一个策略是分析 受感染的哺乳动物细胞中的囊泡运输途径。我们发现弓形虫能截获哺乳动物 RAB囊泡与循环、内吞和分泌途径有关，并将这些囊泡隔离到 附着于PV膜上的膜性小管网络。我们的第二种方法是分析非 脂类转移的囊泡途径，特别是膜接触部位(MCS)。通过检查身体状况 哺乳动物宿主细胞器与PV膜的连通性，我们表明弓形虫吸引宿主 ER小管和脂滴连接到PV，在那里它们在一定距离上与PV膜紧密相对 让人想起细胞器间的接触。哺乳动物内质网驻留小泡相关膜蛋白(VAP)， MCS的组成成分与PV膜相关，提示脂质的潜在开发 弓形虫转运蛋白获取脂质。 基于这些初步观察，我们提出了两种弓形虫清除脂质的模型 哺乳动物的囊泡性或非囊泡性脂质运输途径。我们将通过以下方式评估这些模型的步骤 确定参与宿主囊泡途径截获的分子机制和机制 弓形虫(Aim 1)，PV中膜小管网络的形成及其在哺乳动物中的作用 细胞器隔离(AIM 2)与哺乳动物通过非囊泡转移获得脂类 细胞器与PVM密切相关，可能通过MCS(目标3)。 完成这些目标将揭开弓形虫介导的脂质回收过程的复杂性， 提供机械性细节，并确定未来干预的目标。事实上，弓形虫可以导致致命的 免疫受损个体中的脑炎，以及目前弓形虫病的治疗选择有限。 此外，研究弓形虫用来篡夺Rab介导的囊泡运输的机制可能会产生 对Rab GTP酶如何协调哺乳动物细胞的膜运输的有价值的见解。检查 弓形虫开发的利用MCS的潜在策略也可能提供关于如何 MCs的缺失会影响哺乳动物的细胞生理和机体功能。

SUMMARY Lipids are transferred between membranes by vesicular and non-vesicular routes. Many microorganisms that infect mammalian cells subvert the function of these host cellular lipid trafficking pathways to acquire lipids. Toxoplasma gondii is an obligate intracellular parasite that multiplies in the cytoplasm of mammalian cells within a self-made membrane-bound compartment – the parasitophorous vacuole (PV). The PV of T. gondii does not fuse with host organelles. However, we showed that the parasite’s intracellular survival relies on lipids retrieved from various mammalian organelles. For example, T. gondii scavenges cholesterol and sphingolipids from host endocytic organelles and Golgi vesicles, respectively, which raises the perplexing question of how T. gondii can access the lipid content of these organelles without fusion. To address this issue, our first strategy was to analyze vesicular trafficking pathways in infected mammalian cells. We showed that Toxoplasma intercepts mammalian Rab vesicles associated with recycling, endocytic and secretory pathways, and sequesters these vesicles into a network of membranous tubules appended to the PV membrane. Our second approach was to analyze non- vesicular routes of lipid transfer, specifically Membrane Contact Sites (MCS). By examining the physical connectivity of mammalian host organelles with the PV membrane, we showed that Toxoplasma attracts host ER tubules and lipid droplets to the PV, where they are closely apposed to the PV membrane at distances reminiscent of inter-organelle contacts. Mammalian ER-resident Vesicle-Associated Membrane Proteins (VAP), components of MCS, are associated with the PV membrane, suggesting the potential exploitation of Lipid Transfer Proteins by Toxoplasma for lipid acquisition. Based on these preliminary observations, we propose two models for lipid scavenging by Toxoplasma either mammalian vesicular or non-vesicular lipid transport pathways. We will assess the steps of these models by defining the molecular machineries and mechanisms involved in the interception of host vesicular pathways by T. gondii (Aim 1), the formation of a network of membranous tubules in the PV and its role in mammalian organelle sequestration (Aim 2) and the acquisition of lipids via non-vesicular transfer from mammalian organelles closely associated with the PVM, possibly through MCS (Aim 3). Completing these aims would unravel the complexity of lipid salvage processes mediated by Toxoplasma, providing mechanistic details and identifying future targets for intervention. Indeed, T. gondii can cause fatal encephalitis in immunocompromised individuals, and current treatment options for toxoplasmosis are limited. Furthermore, studying the mechanisms used by Toxoplasma to usurp Rab-mediated vesicle trafficking may yield valuable insights into how Rab GTPases coordinate membrane transport in mammalian cells. Examining the potential strategies developed by Toxoplasma to exploit MCS may also provide important information on how the loss of MCS affect mammalian cellular physiology and organismal function.