Mechanisms and functions of host organelle usurpation by intravacuolar Toxoplasma

液泡内弓形虫侵占宿主细胞器的机制和功能

• 批准号: 10649407
• 负责人: Isabelle Coppens
• 金额: $ 69.69万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-20 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10649407
• 关键词: Abbreviations; Address; Affect; Binding; Biochemical; Biochemical Pathway; Biological Assay; Cell membrane; Cell physiology; Cells; Cellular biology; Cholesterol; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cytoplasm; Cytoplasmic Granules; Development; Docking; Encephalitis; Endosomes; Filament; Future; Genes; Genetic Screening; Genetic Techniques; Genome; Goals; Golgi Apparatus; HIV/AIDS; Immunocompromised Host; Immunofluorescence Immunologic; Individual; Infection; Intercept; Intervention; Lipids; Mammalian Cell; Mediating; Membrane; Membrane Proteins; Microscopic; Modeling; Molecular; Nature; Nutrient; Organelles; Parasites; Pathway interactions; Patients; Penetration; Phosphatidylcholine-Sterol O-Acyltransferase; Phospholipase; Plasma Cells; Play; Process; Protein Secretion; Proteins; Proteome; Recycling; Resources; Role; Route; Site; Sorting; Sphingolipids; System; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Transmembrane Transport; Vacuole; Vesicle; Work; chemotherapy; design; differential expression; genetic manipulation; insight; lipid transfer protein; lipid transport; microorganism; model organism; mutant; novel; obligate intracellular parasite; opportunistic pathogen; pathogen; protein complex; rab GTP-Binding Proteins; recruit; response; trafficking; vesicle-associated membrane protein

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.

概括 脂质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破坏这些宿主细胞脂质运输途径的功能以获得脂质。 弓形虫是一种专性细胞内寄生虫，可在哺乳动物细胞的细胞质中繁殖。 自制的膜结合区室——寄生液泡（PV）。弓形虫的 PV 不 与宿主细胞器融合。然而，我们发现寄生虫的细胞内存活依赖于回收的脂质 来自各种哺乳动物细胞器。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体囊泡分别，这提出了一个令人困惑的问题：弓形虫如何 无需融合即可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 受感染哺乳动物细胞中的囊泡运输途径。我们证明弓形虫可以拦截哺乳动物 Rab 囊泡与回收、内吞和分泌途径相关，并将这些囊泡隔离在 附着在 PV 膜上的膜管网络。我们的第二种方法是分析非 脂质转移的囊泡途径，特别是膜接触位点（MCS）。通过检查身体 通过哺乳动物宿主细胞器与 PV 膜的连接，我们发现弓形虫会吸引宿主 ER 小管和脂滴到 PV，它们与 PV 膜相距一定距离 让人想起细胞器间的接触。哺乳动物内质网驻留的囊泡相关膜蛋白 (VAP)， MCS 的成分与 PV 膜相关，表明脂质的潜在开发利用 通过弓形虫转移蛋白质以获取脂质。 基于这些初步观察，我们提出了弓形虫清除脂质的两种模型： 哺乳动物囊泡或非囊泡脂​​质转运途径。我们将通过以下方式评估这些模型的步骤 定义参与拦截宿主囊泡途径的分子机器和机制 T. gondii（目标 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（目标 2）和通过非囊泡转移从哺乳动物获取脂质 与 PVM 密切相关的细胞器，可能通过 MCS（目标 3）。 完成这些目标将揭示弓形虫介导的脂质回收过程的复杂性， 提供机制细节并确定未来的干预目标。事实上，弓形虫可导致致命 免疫功能低下个体的脑炎，目前弓形体病的治疗选择有限。 此外，研究弓形虫侵占 Rab 介导的囊泡运输的机制可能会产生 关于 Rab GTPases 如何协调哺乳动物细胞中的膜运输的宝贵见解。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利用 MCS 的重要信息。 MCS 的丧失会影响哺乳动物的细胞生理学和有机体功能。