A GCN2-like eukaryotic initiation factor 2 kinase increases the viability of extracellular Toxoplasma gondii parasites

Toxoplasmosis is a significant opportunistic infection caused by the protozoan parasite Toxoplasma gondii, an obligate intracellular pathogen that relies on host cell nutrients for parasite proliferation. Toxoplasma parasites divide until they rupture the host cell, at which point the extracellular parasites must survive until they find a new host cell. Recent studies have indicated that phosphorylation of Toxoplasma eukaryotic translation initiation factor 2-alpha (TgIF2α) plays a key role in promoting parasite viability during times of extracellular stress. Here we report the cloning and characterization of a TgIF2α kinase designated TgIF2K-D that is related to GCN2, a eukaryotic initiation factor 2α (eIF2α) kinase known to respond to nutrient starvation in other organisms. TgIF2K-D is present in the cytosol of both intra- and extracellular Toxoplasma parasites and facilitates translational control through TgIF2α phosphorylation in extracellular parasites. We generated a TgIF2K-D knockout parasite and demonstrated that loss of this eIF2α kinase leads to a significant fitness defect that stems from an inability of the parasite to adequately adapt to the environment outside host cells. This phenotype is consistent with that reported for our nonphosphorylatable TgIF2α mutant (S71A substitution), establishing that TgIF2K-D is the primary eIF2α kinase responsible for promoting extracellular viability of Toxoplasma. These studies suggest that eIF2α phosphorylation and translational control are an important mechanism by which vulnerable extracellular parasites protect themselves while searching for a new host cell. Additionally, TgIF2α is phosphorylated when intracellular parasites are deprived of nutrients, but this can occur independently of TgIF2K-D, indicating that this activity can be mediated by a different TgIF2K.     

Autophagy protein Atg3 is essential for maintaining mitochondrial integrity and for normal intracellular development of Toxoplasma gondii tachyzoites

Autophagy is a cellular process that is highly conserved among eukaryotes and permits the degradation of cellular material. Autophagy is involved in multiple survival-promoting processes. It not only facilitates the maintenance of cell homeostasis by degrading long-lived proteins and damaged organelles, but it also plays a role in cell differentiation and cell development. Equally important is its function for survival in stress-related conditions such as recycling of proteins and organelles during nutrient starvation. Protozoan parasites have complex life cycles and face dramatically changing environmental conditions; whether autophagy represents a critical coping mechanism throughout these changes remains poorly documented. To investigate this in Toxoplasma gondii, we have used TgAtg8 as an autophagosome marker and showed that autophagy and the associated cellular machinery are present and functional in the parasite. In extracellular T. gondii tachyzoites, autophagosomes were induced in response to amino acid starvation, but they could also be observed in culture during the normal intracellular development of the parasites. Moreover, we generated a conditional T. gondii mutant lacking the orthologue of Atg3, a key autophagy protein. TgAtg3-depleted parasites were unable to regulate the conjugation of TgAtg8 to the autophagosomal membrane. The mutant parasites also exhibited a pronounced fragmentation of their mitochondrion and a drastic growth phenotype. Overall, our results show that TgAtg3-dependent autophagy might be regulating mitochondrial homeostasis during cell division and is essential for the normal development of T. gondii tachyzoites.     

GCN2-like eIF2α kinase manages the amino acid starvation response in Toxoplasma gondii

The apicomplexan protozoan Toxoplasma gondii is a significant human and veterinary pathogen. As an obligate intracellular parasite, Toxoplasma depends on nutrients provided by the host cell and needs to adapt to limitations in available resources. In mammalian cells, translational regulation via GCN2 phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) is a key mechanism for adapting to nutrient stress. Toxoplasma encodes two GCN2-like protein kinases, TgIF2K-C and TgIF2K-D. We previously showed that TgIF2K-D phosphorylates T. gondii eIF2α (TgIF2α) upon egress from the host cell, which enables the parasite to overcome exposure to the extracellular environment. However, the function of TgIF2K-C remained unresolved. To determine the functions of TgIF2K-C in the parasite, we cloned the cDNA encoding TgIF2K-C and generated knockout parasites of this TgIF2α kinase to study its function during the lytic cycle. The TgIF2K-C knockout did not exhibit a fitness defect compared with parental parasites. However, upon infection of human fibroblasts that were subsequently cultured in glutamine-free medium, the intracellular TgIF2K-C knockout parasites were impeded for induced phosphorylation of TgIF2α and showed a 50% reduction in the number of plaques formed compared with parental parasites. Furthermore, we found that this growth defect in glutamine-free media was phenocopied in parasites expressing only a non-phosphorylatable TgIF2α (TgIF2α-S71A), but not in a TgIF2K-D knockout. These studies suggest that Toxoplasma GCN2-like kinases TgIF2K-C and TgIF2K-D evolved to have distinct roles in adapting to changes in the parasite’s environment.     

Contribution of host lipids to Toxoplasma pathogenesis

As an actively dividing organism, the intracellular parasite Toxoplasma gondii must adjust the size and composition of its membranes in order to accommodate changes due to housekeeping activities, to commit division and in fine to produce new viable progenies. Lipid inventory of T. gondii reveals that the biological membranes of this parasite are composed of a complex mixture of neutral and polar lipids. After examination of the origin of T. gondii membrane lipids, three categories of lipids can be described: (i) lipids scavenged by T. gondii from the host cell; (ii) lipids synthesized in large amounts by the parasite, independently from its host cell; and (iii) lipids produced de novo by the parasite, but whose synthesis does not come close to satisfying the entire parasite's needs. These latter must be adeptly acquired from the host environment. To this end, T. gondii diverts a large variety of lipid precursors from host cytoplasm and efficiently manufacture them into complex lipids. This rather remarkable reliance on host lipid resources for parasite survival opens new avenues to restrict parasite growth. Indeed, parasite starvation can be induced upon deprivation from essential host lipids. Lipid analogues with anti-proliferative properties are voraciously taken up by the parasites, which results in parasite membrane defects, and ultimately death.     

Absence of vacuolar membrane involving Toxoplasma gondii during its intranuclear localization

Tachyzoites of Toxoplasma gondii were located inside the nucleus of both skeletal muscle cells infected in vitro and peritoneal exudate cells collected from infected mouse in vivo. Ultrastructural analysis demonstrated that T. gondii invades the nucleus of host cells by the parasite apical region and with constriction of its body. We noted that the rhoptry, a secretory organelle of the parasite that is involved in the host cell invasion mechanism, was empty in the intranuclear T. gondii. The parasites were found in the nuclear matrix without evidence of the vacuolar membrane. Frequently, new parasites invaded host cell nucleus, which was already infected. The significance of this nuclear invasion could reflect an alternative route of T. gondii for its transitory survival or an escape mechanism from the host immune response during the in vivo infection (or both).     

Analysis of Monensin Sensitivity in Toxoplasma gondii Reveals Autophagy as a Mechanism for Drug Induced Death

Understanding the mechanisms by which anti-parasitic drugs alter the physiology and ultimately kill is an important area of investigation. Development of novel parasitic drugs, as well as the continued utilization of existing drugs in the face of resistant parasite populations, requires such knowledge. Here we show that the anti-coccidial drug monensin kills Toxoplasma gondii by inducing autophagy in the parasites, a novel mechanism of cell death in response to an antimicrobial drug. Monensin treatment results autophagy, as shown by translocation of ATG8 to autophagosomes, as well as causing marked morphological changes in the parasites' mitochondria. Use of the autophagy inhibitor 3-methyladenine blocks autophagy and mitochondrial alterations, and enhances parasite survival, in monensin-exposed parasites, although it does not block other monensin-induced effects on the parasites, such as late S-phase cell cycle arrest. Monensin does not induce autophagy in a parasite strain deficient in the mitochondrial DNA repair enzyme TgMSH-1 an enzyme that mediates monensin-induced late S-phase arrest. TgMSH-1 therefore either mediates cell cycle arrest and autophagy independently, or autophagy occurs downstream of cell cycle arrest in a manner analogous to apoptosis of cells arrested in G(2) of the cell cycle. Overall, our results point to autophagy as a potentially important mode of cell death of protozoan parasites in response to antimicrobial drugs and indicate that disruption of the autophagy pathway could result in drug resistance.     

Apoptotic-like Leishmania exploit the host′s autophagy machinery to reduce T-cell-mediated parasite elimination

Apoptosis is a well-defined cellular process in which a cell dies, characterized by cell shrinkage and DNA fragmentation. In parasites like Leishmania, the process of apoptosis-like cell death has been described. Moreover upon infection, the apoptotic-like population is essential for disease development, in part by silencing host phagocytes. Nevertheless, the exact mechanism of how apoptosis in unicellular organisms may support infectivity remains unclear. Therefore we investigated the fate of apoptotic-like Leishmania parasites in human host macrophages. Our data showed—in contrast to viable parasites—that apoptotic-like parasites enter an LC3⁺, autophagy-like compartment. The compartment was found to consist of a single lipid bilayer, typical for LC3-associated phagocytosis (LAP). As LAP can provoke anti-inflammatory responses and autophagy modulates antigen presentation, we analyzed how the presence of apoptotic-like parasites affected the adaptive immune response. Macrophages infected with viable Leishmania induced proliferation of CD4⁺ T-cells, leading to a reduced intracellular parasite survival. Remarkably, the presence of apoptotic-like parasites in the inoculum significantly reduced T-cell proliferation. Chemical induction of autophagy in human monocyte-derived macrophage (hMDM), infected with viable parasites only, had an even stronger proliferation-reducing effect, indicating that host cell autophagy and not parasite viability limits the T-cell response and enhances parasite survival. Concluding, our data suggest that apoptotic-like Leishmania hijack the host cells´ autophagy machinery to reduce T-cell proliferation. Furthermore, the overall population survival is guaranteed, explaining the benefit of apoptosis-like cell death in a single-celled parasite and defining the host autophagy pathway as a potential therapeutic target in treating Leishmaniasis.     

Toxoplasma gondii lacks the enzymes required for de novo arginine biosynthesis and arginine starvation triggers cyst formation

Two separate carbamoyl phosphate synthetase activities are required for the de novo synthesis of pyrimidines and arginine in most eukaryotes. Toxoplasma gondii is novel in possessing a single carbamoyl phosphate synthetase II gene that corresponds to a glutamine-dependent form required for pyrimidine biosynthesis. We therefore examined arginine acquisition in T. gondii to determine whether the single carbamoyl phosphate synthetase II activity could provide both pyrimidine and arginine biosynthesis. We found that arginine deprivation efficiently blocks the replication of intracellular T. gondii, yet has little effect on long-term parasite viability. Addition of citrulline, but not ornithine, rescues the growth defect observed in the absence of exogenous arginine. This rescue with citrulline is ablated when parasites are cultured in a human citrullinemia fibroblast cell line that is deficient in argininosuccinate synthetase activity. These results reveal the absence of genes and activities of the arginine biosynthetic pathway and demonstrate that T. gondii is an arginine auxotroph. Arginine starvation was also found to efficiently trigger differentiation of replicative tachyzoites into bradyzoites contained within stable cyst-like structures. These same parasites expressing bradyzoite antigens can be efficiently switched back to rapidly proliferating tachyzoites several weeks after arginine starvation. We hypothesise that the absence of gene activities that are essential for the biosynthesis of arginine from carbamoyl phosphate confers a selective advantage by increasing bradyzoite switching during the host response to T. gondii infection. These findings are consistent with a model of host-parasite evolution that allowed host control of bradyzoite induction by trading off virulence for increased transmission.     

Conditional expression of Toxoplasma gondii apical membrane antigen-1 (TgAMA1) demonstrates that TgAMA1 plays a critical role in host cell invasion

Toxoplasma gondii is an obligate intracellular parasite and an important human pathogen. Relatively little is known about the proteins that orchestrate host cell invasion by T. gondii or related apicomplexan parasites (including Plasmodium spp., which cause malaria), due to the difficulty of studying essential genes in these organisms. We have used a recently developed regulatable promoter to create a conditional knockout of T. gondii apical membrane antigen-1 (TgAMA1). TgAMA1 is a transmembrane protein that localizes to the parasite's micronemes, secretory organelles that discharge during invasion. AMA1 proteins are conserved among apicomplexan parasites and are of intense interest as malaria vaccine candidates. We show here that T. gondii tachyzoites depleted of TgAMA1 are severely compromised in their ability to invade host cells, providing direct genetic evidence that AMA1 functions during invasion. The TgAMA1 deficiency has no effect on microneme secretion or initial attachment of the parasite to the host cell, but it does inhibit secretion of the rhoptries, organelles whose discharge is coupled to active host cell penetration. The data suggest a model in which attachment of the parasite to the host cell occurs in two distinct stages, the second of which requires TgAMA1 and is involved in regulating rhoptry secretion.     

Antimonial-induced increase in intracellular Ca2+ through non-selective cation channels in the host and the parasite is responsible for apoptosis of intracellular Leishmania donovani amastigotes

The capability of the obligate intracellular parasites like Leishmania donovani to survive within the host cell parasitophorous vacuoles as nonmotile amastigotes determines disease pathogenesis, but the mechanism of elimination of the parasites from these vacuoles are not well understood. By using the anti-leishmanial drug potassium antimony tartrate, we demonstrate that, upon drug exposure, intracellular L. donovani amastigotes undergo apoptotic death characterized by nuclear DNA fragmentation and externalization of phosphatidylserine. Changes upstream of DNA fragmentation included generation of reactive oxygen species like superoxide, nitric oxide, and hydrogen peroxide that were primarily concentrated in the parasitophorous vacuoles. In the presence of antioxidants like N-acetylcysteine or Mn(III) tetrakis(4-benzoic acid)porphyrin chloride, an inhibitor of inducible nitric-oxide synthase, a diminution of reactive oxygen species generation and improvement of amastigote survival were observed, suggesting a close link between drug-induced oxidative stress and amastigote death. Changes downstream to reactive oxygen species increase involved elevation of intracellular Ca2+ concentrations in both the parasite and the host that was preventable by antioxidants. Flufenamic acid, a non-selective cation channel blocker, decreased the elevation of Ca2+ in both the cell types and reduced amastigote death, thus establishing a central role of Ca2+ in intracellular parasite clearance. This influx of Ca2+ was preceded by a fall in the amastigote mitochondrial membrane potential. Therefore, this study projects the importance of flufenamic acid-sensitive non-selective cation channels as important modulators of antimonial efficacy and lends credence to the suggestion that, within the host cell, apoptosis is the preferred mode of death for the parasites.     

In vivo and in vitro Leishmania amazonensis infection induces autophagy in macrophages

Autophagy is the primary mechanism of degradation of cellular proteins and at least two functions can be attributed to this biological phenomenon: increased nutrient supply via recycling of the products of autophagy under nutrient starvation; and antimicrobial response involved in the innate immune system. Many microorganisms induce host cell autophagy and it has been proposed as a pathway by which parasites compete with the host cell for limited resources. In this report we provide evidence that the intracellular parasite Leishmania amazonensis induces autophagy in macrophages. Using western blotting, the LC3II protein, a marker of autophagosomes, was detected in cell cultures with a high infection index. Macrophages infected with L. amazonensis were examined by transmission electronic microscopy, which revealed enlarged myelin-like structures typical late autophagosome and autolysosome. Other evidence indicating autophagy was Lysotracker red dye uptake by the macrophages. Autophagy also occurs in the leishmaniasis skin lesions of BALB/c mice, detected by immunohistochemistry with anti-LC3II antibody. In this study, autophagy inhibitor 3-methyladenine (3MA) reduced the infection index, while autophagy inductors, such as rapamycin or starvation, did not alter the infection index in cultivated macrophages, suggesting that one aspect of the role of autophagy could be the provision of nutritive support to the parasite.     

The phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii reveal unusual adaptations within and beyond the parasites' boundaries

Plasmodium falciparum and Toxoplasma gondii are obligate intracellular apicomplexan parasites that rapidly invade and extensively modify host cells. Protein phosphorylation is one mechanism by which these parasites can control such processes. Here we present a phosphoproteome analysis of peptides enriched from schizont stage P. falciparum and T. gondii tachyzoites that are either "intracellular" or purified away from host material. Using liquid chromatography-tandem mass spectrometry, we identified over 5,000 and 10,000 previously unknown phosphorylation sites in P. falciparum and T. gondii, respectively, revealing that protein phosphorylation is an extensively used regulation mechanism both within and beyond parasite boundaries. Unexpectedly, both parasites have phosphorylated tyrosines, and P. falciparum has unusual phosphorylation motifs that are apparently shaped by its A:T-rich genome. This data set provides important information on the role of phosphorylation in the host-pathogen interaction and clues to the evolutionary forces operating on protein phosphorylation motifs in both parasites.     

Direct inhibition of cytochrome c-induced caspase activation in vitro by Toxoplasma gondii reveals novel mechanisms of interference with host cell apoptosis

The intracellular parasite Toxoplasma gondii is known to inhibit apoptosis of its host cell. The molecular mechanisms of this interference are, however, not yet completely understood. We show here that viable parasites prominently inhibited the activation of caspase 3/7 induced by cytochrome c, dATP and dithiothreitol in cytosolic extracts of human-derived Jurkat leukemic T cells. In contrast, granzyme B-induced caspase activity was only slightly diminished. De novo protein biosynthesis by T. gondii was dispensable for the inhibition of cytochrome c-induced caspase activation. Furthermore, a complete parasite lysate or, more importantly, molecules released by extracellular parasites mediated the interaction with the caspase cascade. The cell-free system applied here is thus a valuable tool to study the interaction of T. gondii and possibly other intracellular pathogens with host cell apoptosis.     

Death receptor ligation or exposure to perforin trigger rapid egress of the intracellular parasite Toxoplasma gondii

The obligate intracellular parasite Toxoplasma gondii chronically infects up to one-third of the global population, can result in severe disease in immunocompromised individuals, and can be teratogenic. In this study, we demonstrate that death receptor ligation in T. gondii-infected cells leads to rapid egress of infectious parasites and lytic necrosis of the host cell, an active process mediated through the release of intracellular calcium as a consequence of caspase activation early in the apoptotic cascade. Upon acting on infected cells via death receptor- or perforin-dependent pathways, T cells induce rapid egress of infectious parasites able to infect surrounding cells, including the Ag-specific effector cells.     

Detection of a novel parasite kinase activity at the Toxoplasma gondii parasitophorous vacuole membrane capable of phosphorylating host IkappaBalpha

Toxoplasma gondii activates the NF-kappaB pathway in the infected host cell resulting in upregulation of pro-survival genes and prevention of apoptosis. Manipulation of the NF-kappaB cascade by T. gondii correlates with the localization of phosphorylated IkappaB at the parasitophorous vacuole membrane (PVM). This suggests a parasite-mediated event, involving the recruitment and activation of the host IkappaB kinase (IKK) complex, as has been observed with the related protozoan Theileria parva. In contrast to Theileria, confocal microscopy studies showed no apparent hijacking of IKKalpha, IKKbeta, or their activated phosphorylated forms at the T. gondii PVM. Remarkably, phosphorylation of IkappaBalpha at Ser 32/36 was observed at the PVM of T. gondii-infected IKKalpha-/-, IKKbeta-/- and IKKalpha/beta double-knockout (IKKalpha/beta-/-) fibroblasts, suggesting the involvement of a parasite kinase activity independent of host IKK. The presence of a putative T. gondii IkappaB kinase was examined by in vitro kinase assays using GST-IkappaBalpha constructs and protein extracts from both extracellular parasites and PVM fractions. Interestingly, an activity capable of phosphorylating IkappaBalpha at the critical Ser 32/36 sites was identified in parasite extracts, a property restricted to the IKK signalosome. Taken together, our data support the role for a T. gondii kinase involved in phosphorylation of host cell IkappaBalpha and suggest an unusual mechanism utilized by an intracellular pathogen capable of manipulating the NF-kappaB pathway.     

Role of autophagy in the host defense against Toxoplasma gondii in astrocytes

Autophagy has recently been implicated in the host defense against the intracellular protozoan pathogen, Toxoplasma gondii, a major opportunistic pathogen of the central nervous system in immunosuppressed individuals. In both IFNγ-activated macrophages and astrocytes, the p47 GTPases traffic to the T. gondii parasitophorous vacuole, followed by vacuolar disruption, parasite killing, and clearance of the dead parasites. In macrophages, it is relatively well established that autophagy is involved in parasite elimination and killing. The role of autophagy in parasite elimination in astrocytes, a dominant host cell in the central nervous system, is much less clear. Our studies indicate that in IFNγ-stimulated astrocytes, autophagy of disrupted vacuoles and/or dead parasites does not occur but rather that degradation of the parasite occurs in the host cytoplasm. However, recent studies indicate autophagy may be involved in the elimination of the degraded parasite material from the astrocyte host cell cytoplasm and suggest that autophagous removal of degraded parasite material may be necessary for survival of the host cell. Delivery of parasite antigen from the cytosol to the endolysosomal compartments in astrocytes is of importance as it suggests a pathway by which astrocytes could present Toxoplasma antigens via the MHC Class II pathway and function as an antigen-presenting cell for the parasite in the brain.     

Hydroxyurea inhibits intracellular Toxoplasma gondii multiplication

Tachyzoites of Toxoplasma gondii multiply within the parasitophorous vacuole (PV) until the lysis of the host cell. This study was undertaken to evaluate the effect of hydroxyurea (a specific drug that arrests cell division at G1/S phase) on the multiplication of T. gondii tachyzoites in infected Vero cells. Infected host cells were treated with hydroxyurea for periods varying from 5 to 48 h, and the survival and morphology of the parasite were determined. Hydroxyurea arrested intracellular T. gondii multiplication in all periods tested. After 48 h of incubation with hydroxyurea, intracellular parasites were not easily observed in Vero cells. Ultrastructural observations showed that infected host cells treated with hydroxyurea for 24 h or more presented disrupted intracellular parasites within the PV. However, the host cells exhibited a normal morphology. Our observations suggest that hydroxyurea was able to interfere with the cycle of the intracellular parasite, leading to the complete destruction of the T. gondii without affecting the host cells.     

Autophagic elimination of intracellular parasites: convergent induction by IFN-gamma and CD40 ligation?

Autophagy has recently been implicated in the immune elimination of the intracellular protozoan parasite, Toxoplasma gondii. Toxoplasma and other apicomplexan parasites actively invade host cells and form nonfusogenic parasitophorous vacuoles. Nevertheless, following entry into IFN-gamma-activated effector macrophages, vesiculation of the parasite vacuole or PV membrane ensues, in a process dependent upon the activity of p47 GTPases induced by IFN-gamma signaling. Subsequent disruption of the plasma membrane of the stripped parasites precedes autophagolysosomal elimination of T. gondii. In contrast, ligation of the CD40 receptor and autocrine signaling by TNF activate a seemingly distinct, p47 GTPase-independent mechanism leading to autophagic elimination of intracellular T. gondii, without prior disruption of the pathogen vacuole. Thus, two key pathways of the cell-mediated immune response, namely IFN-gamma and CD40/CD40L, trigger a common autophagolysosomal endpoint of parasite elimination, via distinct intermediary mechanisms.     

Microneme rhomboid protease TgROM1 is required for efficient intracellular growth of Toxoplasma gondii

Rhomboids are serine proteases that cleave their substrates within the transmembrane domain. Toxoplasma gondii contains six rhomboids that are expressed in different life cycle stages and localized to different cellular compartments. Toxoplasma rhomboid protein 1 (TgROM1) has previously been shown to be active in vitro, and the orthologue in Plasmodium falciparum processes the essential microneme protein AMA1 in a heterologous system. We investigated the role of TgROM1 to determine its role during in vitro growth of T. gondii. TgROM1 was localized in the secretory pathway of the parasite, including the Golgi apparatus and micronemes, which contain adhesive proteins involved in invasion of host cells. However, unlike other micronemal proteins, TgROM1 was not released onto the parasite surface during cell invasion, suggesting it does not play a critical role in cell invasion. Suppression of TgROM1 using the tetracycline-regulatable system revealed that ROM1-deficient parasites were outcompeted by wild-type T. gondii. ROM1-deficient parasites showed only modest decrease in invasion but replicated more slowly than wild-type cells. Collectively, these results indicate that ROM1 is required for efficient intracellular growth by T. gondii.     

Role of ATG3 in the parasite Toxoplasma gondii: Autophagy in an early branching eukaryote

Toxoplasma gondii belongs to the phylum Apicomplexa, a diverse group of early branching unicellular eukaryotes related to dinoflagellates and ciliates. Like several other Apicomplexa such as Plasmodium (the causative agent of malaria), T. gondii is a human pathogen responsible for a potentially lethal disease called toxoplasmosis. Most Apicomplexa have complex life cycles, involving intermediate hosts and vectors, which include obligatory intracellular developmental stages. In the case of malaria and toxoplasmosis, it is that replicative process, leading to the ultimate lysis of the host cell, which is causing the symptoms of the disease. For Toxoplasma, the invasive and fast-replicating form of the parasite is called the tachyzoite. While autophagy has been a fast-growing field of research in recent years, not much was known about the relevance of this catabolic process in medically important apicomplexan parasites. Vesicles resembling autophagosomes had been described in drug-treated Plasmodium parasites in the early 1970s and a potential role for autophagy in organelle recycling during differentiation between Plasmodium life stages has also been recently described. Interestingly, recent database searches have identified putative orthologs of the core machinery responsible for the formation of autophagosomes in several protists, including Toxoplasma. In spite of an apparently reduced machinery (only about one-third of the yeast ATG genes appear to be conserved), T. gondii seemed thus able to perform macroautophagy, but the cellular functions of the pathway for this parasite remained to be demonstrated.