SummaryThe intracellular protozoan Toxoplasma gondii lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this essential lipid from the host environment. In this study, we demonstrated that T. gondii diverts cholesterol from low-density lipoproteins for cholesteryl ester synthesis and storage in lipid bodies. We identified and characterized two isoforms of acyl-CoA:cholesterol acyltransferase

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Summary The role of endocytosis in nutrient uptake by Toxoplasma gondii is unknown. To explore this issue, we characterized an endosomal compartment by identifying a T. gondii Rab5 homologue, a molecular marker for early endosomes in eukaryotic cells. The deduced amino acid sequence of the T. gondii Rab5 gene encodes a protein of 240 amino acids, which we termed TgRab51. TgRab51 was epitope‐tagged at the N‐terminus, expressed in the parasite, and localized by immunofluorescence and immunoelectron microscopy to tubulovesicular structures anterior to the parasite nucleus and adjacent to, but distinct from the Golgi. By immunofluorescence analysis, TgRab51wt‐HA staining partially overlapped with Golgi/TGN markers, but not with the T. gondii secretory organelles. A dominant positive mutant, TgRab51Q103L‐HA, enhanced uptake of exogenous cholesterol analogues in intracellular parasites, augmented formation of lipid droplets and accelerated parasite growth. Brefeldin A disrupted the TgRab51 compartment, and altered the distribution of fluorescent exogenous cholesterol in cells expressing TgRab51Q103L‐HA. These results suggest that TgRab51 facilitates sterol uptake, possibly through a Golgi‐dependent pathway.

Chemotherapy of apicomplexan parasites is limited by emerging drug resistance or lack of novel targets. PfHT1, the Plasmodium falciparum hexose transporter 1, is a promising new drug target because asexual-stage malarial parasites depend wholly on glucose for energy. We have performed a comparative functional characterization of PfHT1 and hexose transporters of the simian malarial parasite P. knowlesi (PkHT1), the rodent parasite P. yoelii (PyHT1) and the human apicomplexan parasite Toxoplasma gondii ( T. gondii glucose transporter 1, TgGT1). PkHT1 and PyHT1 share >70% amino acid identity with PfHT1, while TgGT1 is more divergent (37.2% identity). All transporters mediate uptake of D-glucose and D-fructose. PyHT1 has an affinity for glucose ( K (m) approximately 0.12 mM) that is higher than that for PkHT1 ( K (m) approximately 0.67 mM) or PfHT1 ( K (m) approximately 1 mM). TgGT1 is highly temperature dependent (the Q (10) value, the fold change in activity for a 10 degrees C change in temperature, was >7) compared with Plasmodium transporters ( Q (10), 1.5-2.5), and overall has the highest affinity for glucose ( K (m) approximately 30 microM). Using active analogues in competition for glucose uptake, experiments show that hydroxyl groups at the C-3, C-4 and C-6 positions are important in interacting with PkHT1, PyHT1 and TgGT1. This study defines models useful to study the biology of apicomplexan hexose permeation pathways, as well as contributing to drug development.

Toxoplasma gondii relies on protein secretion from specialized organelles for invasion of host cells and establishment of a parasitophorous vacuole. We identify T. gondii Rab6 as a regulator of protein transport between post-Golgi dense granule organelles and the Golgi. Toxoplasma Rab6 was localized to cisternal rims of the late Golgi and trans-Golgi network, associated transport vesicles, and microdomains of dense granule and endosomal membranes. Overexpression of wildtype Rab6 or GTP-activated Rab6(Q70L) rerouted soluble dense granule secretory proteins to the Golgi and endoplasmic reticulum and augmented the effect of brefeldin A on Golgi resorption to the endoplasmic reticulum. Parasites expressing a nucleotide-free (Rab6(N124I)) or a GDP-bound (Rab6(T25N)) mutant accumulated dense granule proteins in the Golgi and associated transport vesicles and displayed reduced secretion of GRA4 and a delay in glycosylation of GRA2. Activated Rab6 on Golgi membranes colocalized with centrin during mitosis, and parasite clones expressing Rab6 mutants displayed a partial shift in cytokinesis from endodyogeny (formation of two daughter cells) to endopolygeny (multiple daughter cells). We propose that Toxoplasma Rab6 regulates retrograde transport from post-Golgi secretory granules to the parasite Golgi.

Summary Toxoplasma is a protozoan parasite proficiently adapted to thrive in a parasitophorous vacuole (PV) formed in the cytoplasm of a large variety of mammalian cells. As an actively dividing organism, the parasite must adjust the lipid composition of its membranes to preserve organelle vitality and expand the size of the PV membrane to accommodate growing progeny. We showed that Toxoplasma takes up host lipids and can expel major lipids in an ATP-dependent process. In order to provide detailed mechanistic insights into lipid trafficking phenomena relevant to Toxoplasma biology, we characterized six parasite ATP-binding cassette (ABC) G family transporters and investigated their potential contribution to lipid homeostatic processes. All these transporters are expressed in the parasite and five of them are upregulated upon exposure to sterols. Four ABCG are localized to secretory organelles and the plasma membrane, and promote cholesterol and phospholipid efflux, reflecting the importance in exportation of large amounts of lipids into the PV. Interestingly, one ABCG that is associated with vesicles in the PV and the plasma membrane acts as a cholesterol importer. This last finding expands our current view on the role of some ABCG transporters in eukaryotic sterol influx.

Long after their discovery, the function and biogenesis of rhoptries remain enigmatic. In Apicomplexan parasites, these organelles discharge and their contents are exocytosed at the time of host cell invasion, and are thus proposed to play an essential role in establishing the parasitophorous vacuole. In Toxoplasma gondii, ROP2 is suspected to serve as the molecular link between host cell mitochondria and parasitophorous vacuole membrane. In this study we addressed the function of ROP2. Targeted depletion of ROP2 using a ribozyme-modified antisense RNA strategy resulted in multiple effects on parasite morphology because of a disruption in the formation of mature rhoptries, and an arrest in cytokinesis. The association of host cell mitochondria with the parasitophorous vacuole membrane was abolished and the ROP2-deficient parasites had a reduced uptake of sterol from the host cell. Furthermore, these parasites invaded human fibroblasts poorly and had markedly attenuated virulence in mice. We conclude that rhoptry discharge, and in particular release of ROP2, are essential for parasite invasion, replication and host cell-parasite interaction.

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