Timothy T. Stedman

Host cell lipids control cholesteryl ester synthesis and storage in intracellular Toxoplasma

The 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 (ACAT)‐related enzymes, designated TgACAT1α and TgACAT1β in T. gondii. Both proteins are coexpressed in the parasite, localized to the endoplasmic reticulum and participate in cholesteryl ester synthesis. In contrast to mammalian ACAT, TgACAT1α and TgACAT1β preferentially incorporate palmitate into cholesteryl esters and present a broad sterol substrate affinity. Mammalian ACAT‐deficient cells transfected with either TgACAT1α or TgACAT1β are restored in their capability of cholesterol esterification. TgACAT1α produces steryl esters and forms lipid bodies after transformation in a Saccharomyces cerevisiae mutant strain lacking neutral lipids. In addition to their role as ACAT substrates, host fatty acids and low‐density lipoproteins directly serve as Toxoplasma ACAT activators by stimulating cholesteryl ester synthesis and lipid droplet biogenesis. Free fatty acids significantly increase TgACAT1α mRNA levels. Selected cholesterol esterification inhibitors impair parasite growth by rapid disruption of plasma membrane. Altogether, these studies indicate that host lipids govern neutral lipid synthesis in Toxoplasma and that interference with mechanisms of host lipid storage is detrimental to parasite survival in mammalian cells.

Pleiotropic effect due to targeted depletion of secretory rhoptry protein ROP2 in Toxoplasma gondii.

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.

Toxoplasma gondii Rab5 enhances cholesterol acquisition from host cells

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.

Comparative characterization of hexose transporters of Plasmodium knowlesi, Plasmodium yoelii and Toxoplasma gondii highlights functional differences within the apicomplexan family.

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 Rab6 Mediates a Retrograde Pathway for Sorting of Constitutively Secreted Proteins to the Golgi Complex

Toxoplasma gondii relies on protein secretion from specialized organelles for invasion of host cells and establishment of a parasitophorous vacuole. We identifyT. 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 wild-type 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.

Endocytosis in different lifestyles of protozoan parasitism: role in nutrient uptake with special reference to Toxoplasma gondii.

A fundamental property of any eukaryotic cell is endocytosis, that is the ability to take up external fluid, solutes and particulate matter into membrane-bound intracellular vesicles by various mechanisms. Toxoplasma gondii is an intracellular protozoan parasite of the phylum Apicomplexa with a wide geographical and host range distribution. Significant progress in studying the cell biology of this parasite has been accomplished over the last few years. Only recently endocytic compartments and endocytic trafficking have come to a closer dissection in T. gondii. In this review, we discuss the evidence for an endocytic compartment and present a model for an endocytic pathway in Toxoplasma against a background of endocytosis in kinetoplastida and the extensive insights gained from mammalian and yeast cells.

Cytoplasmic localization of calcium/calmodulin‐dependent protein kinase I‐α depends on a nuclear export signal in its regulatory domain

Calcium/calmodulin‐dependent protein kinase I‐α (CaMKI‐α) is a ubiquitous cytosolic enzyme that phosphorylates a number of nuclear proteins in vitro and has been implicated in transcriptional regulation. We report that cytoplasmic localization of CaMKI‐α depends on CRM1‐mediated nuclear export mediated through a Rev‐like nuclear export signal in the CaMKI‐α regulatory domain. Interaction of CaMKI‐α with a CRM1 complex in vitro is enhanced by incubation with calcium/calmodulin. Translocation of CaMKI‐α into the nucleus involves a conserved sequence located within the catalytic core. Mutation of this sequence partially blocks nuclear entry of an export‐impaired mutant of CaMKI‐α.

Novel roles for ATP‐binding cassette G transporters in lipid redistribution in Toxoplasma

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.

Toxoplasma gondii ADP-ribosylation Factor 1 Mediates Enhanced Release of Constitutively Secreted Dense Granule Proteins

Toxoplasma gondii dense granules are morphologically similar to dense matrix granules in specialized secretory cells, yet are secreted in a constitutive, calcium-independent fashion. We previously demonstrated that secretion of dense granule proteins in permeabilized parasites was augmented by the non-hydrolyzable GTP analogue guanosine 5′-3-O-(thio)triphosphate (GTPγS) (Chaturvedi, S., Qi, H., Coleman, D. L., Hanson, P., Rodriguez, A., and Joiner, K. A. (1998) J. Biol. Chem. 274, 2424–2431). As now demonstrated by pharmacological and electron microscopic approaches, GTPγS enhanced release of dense granule proteins in the permeabilized cell system. To investigate the role of ADP-ribosylation factor 1 (ARF1) in this process, a cDNA encoding T. gondii ARF1 (TgARF1) was isolated. Endogenous and transgenic TgARF1 localized to the Golgi of T. gondii, but not to dense granules. An epitope-tagged mutant of TgARF1 predicted to be impaired in GTP hydrolysis (Q71L) partially dispersed the Golgi signal, with localization to scattered vesicles, whereas a mutant impaired in nucleotide binding (T31N) was cytosolic in location. Both mutants caused partial dispersion of a Golgi/trans-Golgi network marker. TgARF1 mutants inhibited delivery of the secretory reporter,Escherichia coli alkaline phosphatase, to dense granules, precluding an in vivo assessment of the role of TgARF1 in release of intact dense granules. To circumvent this limitation, recombinant TgARF1 was purified using two separate approaches, and used in the permeabilized cell assay. TgARF1 protein purified on a Cibacron G3 column and able to bind GTP stimulated dense granule secretion in the permeabilized cell secretion assay. These results are the first to show that ARF1 can augment release of constitutively secreted vesicles at the target membrane.

En route to the vacuole: Tracing the secretory pathway of Toxoplasma gondii

Publisher Summary This chapter focuses mainly on the highly invasive tachyzoite form of the organism with some mention of events in Toxoplasma gondii bradyzoites. While it is a reasonable assumption that specific nutrients needed by the parasite are not available in either the correct form or in sufficient concentration extracellularly to support growth outside cells and that secreted dense granule proteins and the intravacuolar network assist with nutrient acquisition, the understanding of this issue remains limited at best. Secreted dense granule proteins could instead, or in addition, have roles in promoting or blocking antigen processing, in abrogating components of innate host defense mechanisms, or as structural components. By defining the targeting strategies for secreted dense granule proteins, it will now be possible, even in those circumstances where the gene for the endogenous protein cannot be disrupted, to mis-target copies of the endogenous molecule, looking for potential dominant negative effects. Only after this is done will it be possible to propose functions for Toxoplasma proteins at specific locations within the cell.