Isabelle Coppens

Golgi biogenesis in Toxoplasma gondii

Two models have been put forward to explain the growth of new Golgi during the cell cycle. The first suggests that a new Golgi grows out of the endoplasmic reticulum by de novo synthesis. The second suggests that a pre-existing Golgi is needed for the growth of a new one, that is, the Golgi is an autonomously replicating organelle. To resolve this issue, we have exploited the simplicity of the apicomplexan parasite Toxoplasma gondii, which has only a single Golgi stack. Here we show, by using video fluorescence microscopy and three-dimensional reconstructions of serial thin sections, that the Golgi grows by a process of lateral extension followed by medial fission. Further fission leads to the inheritance by each daughter of a pair of Golgi structures, which then coalesce to re-form a single Golgi. Our results indicate that new Golgi grow by autonomous duplication and raise the possibility that the Golgi is a paired structure that is analogous to centrioles.

Myosin A tail domain interacting protein (MTIP) localizes to the inner membrane complex of Plasmodium sporozoites.

Apicomplexan host cell invasion and gliding motility depend on the parasites actomyosin system located beneath the plasma membrane of invasive stages. Myosin A (MyoA), a class XIV unconventional myosin, is the motor protein. A model has been proposed to explain how the actomyosin motor operates but little is known about the components, topology and connectivity of the motor complex. Using the MyoA neck and tail domain as bait in a yeast two-hybrid screen we identified MTIP, a novel 24 kDa protein that interacts with MyoA. Deletion analysis shows that the 15 amino-acid C-terminal tail domain of MyoA, rather than the neck domain, specifically interacts with MTIP. In Plasmodium sporozoites MTIP localizes to the inner membrane complex (IMC), where it is found clustered with MyoA. The data support a model for apicomplexan motility and invasion in which the MyoA motor protein is associated via its tail domain with MTIP, immobilizing it at the outer IMC membrane. The head domain of the immobilized MyoA moves actin filaments that, directly or via a bridging protein, connect to the cytoplasmic domain of a transmembrane protein of the TRAP family. The actin/TRAP complex is then redistributed by the stationary MyoA from the anterior to the posterior end of the zoite, leading to its forward movement on a substrate or to penetration of a host cell.

Activation of NF-κB by Toxoplasma gondii correlates with increased expression of antiapoptotic genes and localization of phosphorylated IκB to the parasitophorous vacuole membrane

Mammalian cells infected with Toxoplasma gondii are resistant to apoptosis induced by a variety of stimuli. We have demonstrated that the host transcription factor NF-κB plays a pivotal role in the T.-gondii-mediated blockade of apoptosis because inhibition is lost in cells lacking the p65 (RelA) subunit of NF-κB (p65–/–). In the present study, we examined the effects of T. gondii infection on NF-κB activation and the expression of genes involved in the apoptotic cascade. Infection of wild-type mouse embryonic fibroblasts (MEFs) with T.-gondii-induced nuclear translocation of the p50 and p65 subunits of NF-κB as examined by immunoblotting of nuclear extracts, immunofluorescence and electrophoretic mobility shift assays. A comparison of apoptotic gene expression profiles from wild-type and p65–/– MEFs revealed distinct patterns of induction in response to T. gondii infection. In particular, the differences seen in the Bcl-2 and IAP families are consistent with the antiapoptotic responses observed in the resistant wild-type cells compared with the sensitive p65–/– fibroblasts. Consistent with NF-κB activation, T. gondii infection promoted phosphorylation of the inhibitor IκB. Interestingly, phosphorylated IκB was concentrated on the parasitophorous vacuole membrane (PVM), suggesting a parasite-directed event. Results from this study suggest that activation of NF-κB plays an important role in stimulation of antiapoptotic gene expression by T. gondii. Furthermore, recruitment of phosphorylated IκB to the PVM implies the presence of intrinsic factor(s) in T. gondii that might be used to manipulate the NF-κB signaling pathway in the host to elicit a survival response during infection.

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.

Selective Disruption of Phosphatidylcholine Metabolism of the Intracellular Parasite Toxoplasma gondii Arrests Its Growth

Toxoplasma gondii is an intracellular protozoan parasite capable of causing devastating infections in immunocompromised and immunologically immature individuals. In this report, we demonstrate the relative independence of T. gondii from its host cell for aminoglycerophospholipid synthesis. The parasite can acquire the lipid precursors serine, ethanolamine, and choline from its environment and use them for the synthesis of its major lipids, phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn), and phosphatidylcholine (PtdCho), respectively. Dimethylethanolamine (Etn(Me)2), a choline analog, dramatically interfered with the PtdCho metabolism of T. gondii and caused a marked inhibition of its growth within human foreskin fibroblasts. In tissue culture medium supplemented with 2 mm Etn(Me)2, the parasite-induced lysis of the host cells was dramatically attenuated, and the production of parasites was inhibited by more than 99%. The disruption of parasite growth was paralleled by structural abnormalities in its membranes. In contrast, no negative effect on host cell growth and morphology was observed. The data also reveal that the Etn(Me)2-supplemented parasite had a time-dependent decrease in its PtdCho content and an equivalent increase in phosphatidyldimethylethanolamine, whereas other major lipids, PtdSer, PtdEtn, and PtdIns, remained largely unchanged. Relative to host cells, the parasites incorporated more than 7 times as much Etn(Me)2 into their phospholipid. These findings reveal that Etn(Me)2 selectively alters parasite lipid metabolism and demonstrate how selective inhibition of PtdCho synthesis is a powerful approach to arresting parasite growth.

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.

The Plasmodium falciparum Vps4 homolog mediates multivesicular body formation

Members of the apicomplexan family of parasites contain morphologically unique secretory organelles termed rhoptries that are essential for host cell invasion. Rhoptries contain internal membranes, and thus resemble multivesicular bodies. To determine whether multivesicular body endosomal intermediates are formed in Apicomplexa, we used the Plasmodium falciparum homolog of the class E gene, Vps4, as a probe. Endogenous P. falciparum Vps4 (PfVps4) localized to the cytoplasm of P. falciparum trophozoites, and transgenic PfVps4 localized to the cytosol in P. falciparum, in the related parasite Toxoplasma gondii and in COS cells. When mutated to block ATP hydrolysis, transiently expressed PfVps4 localized instead to large vesicular structures in P. falciparum. The same construct, and another mutant blocked in ATP binding, generated large cholesterol-enriched multivesicular bodies in both COS cells and T. gondii. Mutant PfVps4 structures in T. gondii co-localized with markers for early endosomes. These results demonstrate a conservation of Vps4 function across wide phylogenetic boundaries, and indicate that endosomal multivesicular bodies form in both P. falciparum and T. gondii.

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.