Evidence for Host Cells as the Major Contributor of Lipids in the Intravacuolar Network of Toxoplasma-Infected Cells

Abstract

The intracellular parasite Toxoplasma gondii develops inside a parasitophorous vacuole (PV) that derives from the host cell plasma membrane during invasion. Previous electron micrograph images have shown that the membrane of this vacuole undergoes an extraordinary remodeling with an extensive network of thin tubules and vesicles, the intravacuolar network (IVN), which fills the lumen of the PV. While dense granule proteins, secreted during and after invasion, are the main factors for the organization and tubulation of the network, little is known about the source of lipids used for this remodeling. By selectively labeling host cell or parasite membranes, we uncovered evidence that strongly supports the host cell as the primary, if not exclusive, source of lipids for parasite IVN remodeling. Fluorescence recovery after photobleaching (FRAP) microscopy experiments revealed that lipids are surprisingly dynamic within the parasitophorous vacuole and are continuously exchanged or replenished by the host cell. The results presented here suggest a new model for development of the parasitophorous vacuole whereby the host provides a continuous stream of lipids to support the growth and maturation of the PVM and IVN.Toxoplasma gondii is an apicomplexan parasite that infects almost any warm-blooded animal. This ubiquitous parasite is able to proliferate inside the host cell within a unique and dynamic parasitophorous vacuole (PV). This vacuole is established during invasion initially as an invagination of the host cell plasma membrane that surrounds the invading parasite and then pinches off to create a PV (18). During this process, parasite secretory organelles, called dense granules, discharge specific protein factors (GRAs) that remodel the vacuole into a sheltered niche for the parasite.Toxoplasma's ability to acquire essential nutrients from its environment is fundamental to its intracellular lifestyle. It has been postulated that Toxoplasma gains access to various host metabolites (Ͻ1,300 Da) via diffusion through small pores in the PV membrane (PVM) (14). While no direct connection between the PV and the host vesicular transport system has been observed (13), Toxoplasma incorporates low-density lipoprotein (LDL)-derived cholesterol from host endolysosomes (4). Host mitochondria are also recruited efficiently to the cytosolic face of the PV soon after invasion, allowing Toxoplasma to scavenge host lipoic acid (5, 16).In addition to nutrient acquisition from the host cytosol, Toxoplasma facilitates its intracellular lifestyle by actively remodeling the PV. The purpose of this remodeling and the origin of the lipid constituents it uses have not been established. Electron micrographs of the Toxoplasma PV show membrane-like material within the PV and close to the posterior end of the parasites at very early times during invasion (15). A complex of secreted GRA2/GRA4/GRA6 proteins interacts with this material and seems to be required for its reorganization and tubulation (9, 12). Later during infection, similar thin tubul...

Toxoplasma gondii- skeletal muscle cells interaction increases lipid droplet biogenesis and positively modulates the production of IL-12, IFN-g and PGE2

“…Our results suggest that T. gondii -infected SkMC increases the synthesis of LD as well as their content, providing lipids to the parasite and thus contributing to the growth and maturation of the parasitophorous vacuole, as described in another cellular model by Caffaro et al [38]. Initially using the fluorescent dye Nile Red, it was possible to observe the increased formation of LD within the first hour of T. gondii- SkMC interaction and also the presence of LDs next to the parasite, and at 24 h around the PV.…”

Increased Long Chain acyl-Coa Synthetase Activity and Fatty Acid Import Is Linked to Membrane Synthesis for Development of Picornavirus Replication Organelles

“…To monitor FA import we pulse-labeled PV-infected HeLa cells with a fluorescent fatty acid Bodipy 500/510 C4, 9 (bodipy-FA) which is believed to mimic FA with 18 carbon atoms backbone. This and similar molecules are extensively used in lipid metabolism research and it was previously shown to be rapidly utilized by cellular lipid synthesis machinery and incorporated into phospholipids, triglycerides and other natural lipids [24], [25], [26], [27], [28]. The cells were infected at a multiplicity of 50 PFU/cell to ensure simultaneous development of infection, and bodipy-FA was added for 30 min at 4 hours post infection (h p.i.…”

A Lipolytic Lecithin:Cholesterol Acyltransferase Secreted by Toxoplasma Facilitates Parasite Replication and Egress

“…The IVN, a network of tubules and vesicles, is generated from proteins and lipids secreted by the parasite as well as lipids scavenged from the host cell, including phosphatidylcholine, cholesterol from host low density lipoproteins (LDL), and sphingolipids from the host Golgi (21,(62)(63)(64)(65). The distribution of TgLCAT to the IVN suggests a role for this enzyme in the remodeling of the IVN membranes by producing lysophosphatidylcholine that can shape membranes and change their curvature.…”