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: 10363370
• 负责人: Isabelle Coppens
• 金额: $ 53万
• 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/10363370
• 关键词: AIDS/HIV problem; Abbreviations; Address; Affect; Animal Model; Biochemical Genetics; 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; Filament; Future; Genes; Genetic Screening; Genetic Techniques; Goals; Golgi Apparatus; Immunocompromised Host; Immunofluorescence Immunologic; Individual; Infection; Intercept; Intervention; Light; Lipids; Lysosomes; Mammalian Cell; Mediating; Membrane; Membrane Lipids; Membrane Proteins; Microscopic; Modeling; Molecular; Nature; Nutrient; Organelles; Parasites; Pathway interactions; Patients; Phosphatidylcholine-Sterol O-Acyltransferase; Phospholipase; Plasma Cells; Play; Process; Proteins; Proteome; Recycling; Resources; Role; Route; Site; Sorting - Cell Movement; Sphingolipids; System; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Transmembrane Transport; Vacuole; Vesicle; Work; base; chemotherapy; design; differential expression; genetic manipulation; insight; lipid transfer protein; lipid transport; microorganism; 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 脂质通过囊泡和非腔线路线在膜之间转移。许多微生物 感染哺乳动物细胞颠覆这些宿主细胞脂质运输途径的功能以获取脂质。 弓形虫Gondii是一种强大的细胞内寄生虫，在哺乳动物细胞的细胞质中成倍增加 一个自制的膜结合室 - 寄生虫（PV）。 T. gondii的PV没有 与宿主细胞器融合。但是，我们表明寄生虫的细胞内存活依赖于检索的脂质 来自各种哺乳动物细胞器。例如，T。Gondii清除胆固醇和host的鞘脂 内吞细胞器和高尔基蔬菜分别提出了一个令人困惑的问题。 访问这些细胞器的脂质含量而无需融合。为了解决这个问题，我们的第一个策略是分析 感染的哺乳动物细胞中的囊泡运输途径。我们表明弓形虫拦截哺乳动物 与回收，内吞和秘书途径相关的Rab蔬菜，并将这些蔬菜隔离为 附加在光伏膜上的膜管网络。我们的第二种方法是分析非 脂质转移的囊泡路线，特别是膜接触位点（MCS）。通过检查身体 哺乳动物宿主细胞器与PV膜的连通性，我们表明弓形虫景点宿主 ER小管和脂质液滴到PV，在距离上它们紧密应用于PV膜 让人联想到轨道间触点。哺乳动物ER居民囊泡相关的膜蛋白（VAP），，， MC的组件与PV膜有关，表明脂质的潜在剥削 通过弓形虫转移蛋白质以获取脂质。 基于这些初步观察，我们提出了两个用于脂质清除的模型 哺乳动物囊泡或非二脂脂质转运途径。我们将通过 定义通过通过 T. gondii（AIM 1），PV中膜状管网络的形成及其在哺乳动物中的作用 细胞器隔离（AIM 2）和通过从哺乳动物的非敏传递获得脂质 与PVM密切相关的细胞器，通过MCS可能（AIM 3）。 完成这些目标将揭示由弓形虫介导的脂质打捞过程的复杂性， 提供机械细节并确定未来的干预目标。确实，贡迪链球菌可能导致致命 免疫功能低下的个体中的脑炎和弓形虫病的当前治疗选择受到限制。 此外，研究弓形虫用于篡夺Rab介导的囊泡运输的机制可能会产生 对于Rab GTPases如何协调哺乳动物细胞中的膜转运的有价值的见解。检查 弓形虫开发的利用MCS的潜在策略也可能提供有关如何 MC的丧失会影响哺乳动物的细胞生理和有机功能。