The apicoplast is important for the viability and persistence of Toxoplasma gondii bradyzoites.

Toxoplasma gondii is responsible for toxoplasmosis, a disease that can be serious when contracted during pregnancy, but can also be a threat for immunocompromised individuals. Acute infection is associated with the tachyzoite form that spreads rapidly within the host. However, under stress conditions, some parasites can differentiate into cyst-forming bradyzoites, residing mainly in the central nervous system, retina and muscle. Because this latent form of the parasite is resistant to all currently available treatments, and is central to persistence and transmission of the parasite, specific therapeutic strategies targeting this developmental stage need to be found. T. gondii contains a plastid of endosymbiotic origin called the apicoplast, which is an appealing drug target because it is essential for tachyzoite viability and contains several key metabolic pathways that are largely absent from the mammalian host. Its function in bradyzoites, however, is unknown. Our objective was thus to study the contribution of the apicoplast to the viability and persistence of bradyzoites during chronic toxoplasmosis. We have used complementary strategies based on stage-specific promoters to generate conditional bradyzoite mutants of essential apicoplast genes. Our results show that specifically targeting the apicoplast in both in vitro or in vivo-differentiated bradyzoites leads to a loss of long-term bradyzoite viability, highlighting the importance of this organelle for this developmental stage. This validates the apicoplast as a potential area to look for therapeutic targets in bradyzoites, with the aim to interfere with this currently incurable parasite stage.

Disrupting the plastidic iron-sulfur cluster biogenesis pathway in Toxoplasma gondii has pleiotropic effects irreversibly impacting parasite viability.

Like many other apicomplexan parasites, Toxoplasma gondii contains a plastid harboring key metabolic pathways, including the sulfur utilization factor (SUF) pathway that is involved in the biosynthesis of iron-sulfur clusters. These cofactors are crucial for a variety of proteins involved in important metabolic reactions, potentially including plastidic pathways for the synthesis of isoprenoid and fatty acids. It was shown previously that impairing the NFS2 cysteine desulfurase, involved in the first step of the SUF pathway, leads to an irreversible killing of intracellular parasites. However, the metabolic impact of disrupting the pathway remained unexplored. Here, we generated another mutant of this pathway, deficient in the SUFC ATPase, and investigated in details the phenotypic consequences of TgNFS2 and TgSUFC depletion on the parasites. Our analysis confirms that Toxoplasma SUF mutants are severely and irreversibly impacted in division and membrane homeostasis, and suggests a defect in apicoplast-generated fatty acids. However, we show that increased scavenging from the host or supplementation with exogenous fatty acids do not fully restore parasite growth, suggesting that this is not the primary cause for the demise of the parasites and that other important cellular functions were affected. For instance, we also show that the SUF pathway is key for generating the isoprenoid-derived precursors necessary for the proper targeting of GPI-anchored proteins and for parasite motility. Thus, we conclude plastid-generated iron-sulfur clusters support the functions of proteins involved in several vital downstream cellular pathways, which implies the SUF machinery may be explored for new potential anti-Toxoplasma targets.

Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis and overall fitness in Toxoplasma.

Iron-sulfur (Fe-S) clusters are one of the most ancient and ubiquitous prosthetic groups, and they are required by a variety of proteins involved in important metabolic processes. Apicomplexan parasites have inherited different plastidic and mitochondrial Fe-S clusters biosynthesis pathways through endosymbiosis. We have investigated the relative contributions of these pathways to the fitness of Toxoplasma gondii, an apicomplexan parasite causing disease in humans, by generating specific mutants. Phenotypic analysis and quantitative proteomics allowed us to highlight notable differences in these mutants. Both Fe-S cluster synthesis pathways are necessary for optimal parasite growth in vitro, but their disruption leads to markedly different fates: impairment of the plastidic pathway leads to a loss of the organelle and to parasite death, while disruption of the mitochondrial pathway trigger differentiation into a stress resistance stage. This highlights that otherwise similar biochemical pathways hosted by different sub-cellular compartments can have very different contributions to the biology of the parasites, which is something to consider when exploring novel strategies for therapeutic intervention.

TgZFP2 is a novel zinc finger protein involved in coordinating mitosis and budding in Toxoplasma.

Zinc finger proteins (ZFPs) are one of the most abundant groups of proteins with a wide range of molecular functions. We have characterised a Toxoplasma protein that we named TgZFP2, as it bears a zinc finger domain conserved in eukaryotes. However, this protein has little homology outside this region and contains no other conserved domain that could hint for a particular function. We thus investigated TgZFP2 function by generating a conditional mutant. We showed that depletion of TgZFP2 leads to a drastic arrest in the parasite cell cycle, and complementation assays demonstrated the zinc finger domain is essential for TgZFP2 function. More precisely, whereas replication of the nuclear material is initially essentially unaltered, daughter cell budding is seriously impaired: to a large extent newly formed buds fail to incorporate nuclear material. TgZFP2 is found at the basal complex in extracellular parasites and after invasion, but as the parasites progress into cell division, it relocalises to cytoplasmic punctate structures and, strikingly, accumulates in the pericentrosomal area at the onset of daughter cell elongation. Centrosomes have emerged as major coordinators of the budding and nuclear cycles in Toxoplasma, and our study identifies a novel and important component of this machinery.

TgPL2, a patatin-like phospholipase domain-containing protein, is involved in the maintenance of apicoplast lipids homeostasis in Toxoplasma.

Patatin-like phospholipases are involved in numerous cellular functions, including lipid metabolism and membranes remodeling. The patatin-like catalytic domain, whose phospholipase activity relies on a serine-aspartate dyad and an anion binding box, is widely spread among prokaryotes and eukaryotes. We describe TgPL2, a novel patatin-like phospholipase domain-containing protein from the parasitic protist Toxoplasma gondii. TgPL2 is a large protein, in which the key motifs for enzymatic activity are conserved in the patatin-like domain. Using immunofluorescence assays and immunoelectron microscopy analysis, we have shown that TgPL2 localizes to the apicoplast, a non-photosynthetic plastid found in most apicomplexan parasites. This plastid hosts several important biosynthetic pathways, which makes it an attractive organelle for identifying new potential drug targets. We thus addressed TgPL2 function by generating a conditional knockdown mutant and demonstrated it has an essential contribution for maintaining the integrity of the plastid. In absence of TgPL2, the organelle is rapidly lost and remaining apicoplasts appear enlarged, with an abnormal accumulation of membranous structures, suggesting a defect in lipids homeostasis. More precisely, analyses of lipid content upon TgPL2 depletion suggest this protein is important for maintaining levels of apicoplast-generated fatty acids, and also regulating phosphatidylcholine and lysophosphatidylcholine levels in the parasite.

Toxoplasma gondii autophagy-related protein ATG9 is crucial for the survival of parasites in their host.

Autophagy is a conserved, life-promoting, catabolic process involved in the recycling of nonessential cellular components in response to stress. The parasite Toxoplasma gondii is an early-diverging eukaryote in which part of the autophagy machinery is not exclusively involved in a catabolic process but instead has been repurposed for an original function in organelle inheritance during cell division. This function, depending essentially on protein TgATG8 and its membrane conjugation system, is crucial for parasite survival and prevented an in depth study of autophagy in the mutants generated so far in Toxoplasma. Thus, in order to decipher the primary function of canonical autophagy in the parasites, we generated a cell line deficient for TgATG9, a protein thought to be involved in the early steps of the autophagy process. Although the protein proved to be dispensable for the development of these obligate intracellular parasites in vitro, the absence of TgATG9 led to a reduced ability to sustain prolonged extracellular stress. Importantly, depletion of the protein significantly reduced parasites survival in macrophages and markedly attenuated their virulence in mice. Altogether, this shows TgATG9 is important for the fate of Toxoplasma in immune cells and contributes to the overall virulence of the parasite, possibly through an involvement in a canonical autophagy pathway.

Iron depletion has different consequences on the growth and survival of Toxoplasma gondii strains.

Toxoplasma gondii is an obligate intracellular parasite responsible for a pathology called toxoplasmosis, which primarily affects immunocompromised individuals and developing foetuses. The parasite can scavenge essential nutrients from its host to support its growth and survival. Among them, iron is one of the most important elements needed to sustain basic cellular functions as it is involved in a number of key metabolic processes, including oxygen transport, redox balance, and electron transport. We evaluated the effects of an iron chelator on the development of several parasite strains and found that they differed in their ability to tolerate iron depletion. The growth of parasites usually associated with a model of acute toxoplasmosis was strongly affected by iron depletion, whereas cystogenic strains were less sensitive as they were able to convert into persisting developmental forms that are associated with the chronic form of the disease. Ultrastructural and biochemical characterization of the impact of iron depletion on parasites also highlighted striking changes in both their metabolism and that of the host, with a marked accumulation of lipid droplets and perturbation of lipid homoeostasis. Overall, our study demonstrates that although acute iron depletion has an important effect on the growth of T. gondii, it has a more profound impact on actively dividing parasites, whereas less metabolically active parasite forms may be able to avoid some of the most detrimental consequences.

The Toxoplasma gondii mitochondrial transporter ABCB7L is essential for the biogenesis of cytosolic and nuclear iron-sulfur cluster proteins and cytosolic translation.

Iron-sulfur (Fe-S) clusters are ubiquitous inorganic cofactors required for numerous essential cellular pathways. Since they cannot be scavenged from the environment, Fe-S clusters are synthesized de novo in cellular compartments such as the apicoplast, mitochondrion, and cytosol. The cytosolic Fe-S cluster biosynthesis pathway relies on the transport of an intermediate from the mitochondrial pathway. An ATP-binding cassette (ABC) transporter called ABCB7 is responsible for this role in numerous commonly studied organisms, but its role in the medically important apicomplexan parasites has not yet been studied. Here we identify and characterize a Toxoplasma gondii ABCB7 homolog, which we name ABCB7-like (ABCB7L). Genetic depletion shows that it is essential for parasite growth and that its disruption triggers partial stage conversion. Characterization of the knock-down line highlights a defect in the biogenesis of cytosolic and nuclear Fe-S proteins leading to defects in protein translation and other pathways including DNA and RNA replication and metabolism. Our work provides support for a broad conservation of the connection between mitochondrial and cytosolic pathways in Fe-S cluster biosynthesis and reveals its importance for parasite survival. IMPORTANCE: Iron-sulfur (Fe-S) clusters are inorganic cofactors of proteins that play key roles in numerous essential biological processes, for example, respiration and DNA replication. Cells possess dedicated biosynthetic pathways to assemble Fe-S clusters, including a pathway in the mitochondrion and cytosol. A single transporter, called ABCB7, connects these two pathways, allowing an essential intermediate generated by the mitochondrial pathway to be used in the cytosolic pathway. Cytosolic and nuclear Fe-S proteins are dependent on the mitochondrial pathway, mediated by ABCB7, in numerous organisms studied to date. Here, we study the role of a homolog of ABCB7, which we name ABCB7-like (ABCB7L), in the ubiquitous unicellular apicomplexan parasite Toxoplasma gondii. We generated a depletion mutant of Toxoplasma ABCB7L and showed its importance for parasite fitness. Using comparative quantitative proteomic analysis and experimental validation of the mutants, we show that ABCB7L is required for cytosolic and nuclear, but not mitochondrial, Fe-S protein biogenesis. Our study supports the conservation of a protein homologous to ABCB7 and which has a similar function in apicomplexan parasites and provides insight into an understudied aspect of parasite metabolism.

3-Benzylmenadiones and their Heteroaromatic Analogues Target the Apicoplast of Apicomplexa Parasites: Synthesis and Bioimaging Studies.

The apicoplast is an essential organelle for the viability of apicomplexan parasites Plasmodium falciparum or Toxoplasma gondii, which has been proposed as a suitable drug target for the development of new antiplasmodial drug-candidates. Plasmodione, an antimalarial redox-active lead drug is active at low nM concentrations on several blood stages of Plasmodiumsuch as early rings and gametocytes. Nevertheless, its precise biological targets remain unknown. Here, we described the synthesis and the evaluation of new heteroaromatic analogues of plasmodione, active on asexual blood P. falciparum stages and T. gondii tachyzoites. Using a bioimaging-based analysis, we followed the morphological alterations of T. gondii tachyzoites and revealed a specific loss of the apicoplast upon drug treatment. Lipidomic and fluxomic analyses determined that drug treatment severely impacts apicoplast-hosted FASII activity in T. gondii tachyzoites, further supporting that the apicoplast is a primary target of plasmodione analogues. To follow the drug localization, "clickable" analogues of plasmodione were designed as tools for fluorescence imaging through a Cu(I)-catalyzed azide-alkyne cycloaddition reaction. Short-time incubation of two probes with P. falciparum trophozoites and T. gondii tachyzoites showed that the clicked products localize within, or in the vicinity of, the apicoplast of both Apicomplexa parasites. In P. falciparum, the fluorescence signal was also associated with the mitochondrion, suggesting that bioactivation and activity of plasmodione and related analogues are potentially associated with these two organelles in malaria parasites.

International consensus guidelines for the definition, detection, and interpretation of autophagy-dependent ferroptosis.

Macroautophagy/autophagy is a complex degradation process with a dual role in cell death that is influenced by the cell types that are involved and the stressors they are exposed to. Ferroptosis is an iron-dependent oxidative form of cell death characterized by unrestricted lipid peroxidation in the context of heterogeneous and plastic mechanisms. Recent studies have shed light on the involvement of specific types of autophagy (e.g. ferritinophagy, lipophagy, and clockophagy) in initiating or executing ferroptotic cell death through the selective degradation of anti-injury proteins or organelles. Conversely, other forms of selective autophagy (e.g. reticulophagy and lysophagy) enhance the cellular defense against ferroptotic damage. Dysregulated autophagy-dependent ferroptosis has implications for a diverse range of pathological conditions. This review aims to present an updated definition of autophagy-dependent ferroptosis, discuss influential substrates and receptors, outline experimental methods, and propose guidelines for interpreting the results.Abbreviation: 3-MA:3-methyladenine; 4HNE: 4-hydroxynonenal; ACD: accidentalcell death; ADF: autophagy-dependentferroptosis; ARE: antioxidant response element; BH2:dihydrobiopterin; BH4: tetrahydrobiopterin; BMDMs: bonemarrow-derived macrophages; CMA: chaperone-mediated autophagy; CQ:chloroquine; DAMPs: danger/damage-associated molecular patterns; EMT,epithelial-mesenchymal transition; EPR: electronparamagnetic resonance; ER, endoplasmic reticulum; FRET: Försterresonance energy transfer; GFP: green fluorescent protein;GSH: glutathione;IF: immunofluorescence; IHC: immunohistochemistry; IOP, intraocularpressure; IRI: ischemia-reperfusion injury; LAA: linoleamide alkyne;MDA: malondialdehyde; PGSK: Phen Green™ SK;RCD: regulatedcell death; PUFAs: polyunsaturated fatty acids; RFP: red fluorescentprotein;ROS: reactive oxygen species; TBA: thiobarbituricacid; TBARS: thiobarbituric acid reactive substances; TEM:transmission electron microscopy.

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.

The RON2-AMA1 interaction is a critical step in moving junction-dependent invasion by apicomplexan parasites.

Obligate intracellular Apicomplexa parasites share a unique invasion mechanism involving a tight interaction between the host cell and the parasite surfaces called the moving junction (MJ). The MJ, which is the anchoring structure for the invasion process, is formed by secretion of a macromolecular complex (RON2/4/5/8), derived from secretory organelles called rhoptries, into the host cell membrane. AMA1, a protein secreted from micronemes and associated with the parasite surface during invasion, has been shown in vitro to bind the MJ complex through a direct association with RON2. Here we show that RON2 is inserted as an integral membrane protein in the host cell and, using several interaction assays with native or recombinant proteins, we define the region that binds AMA1. Our studies were performed both in Toxoplasma gondii and Plasmodium falciparum and although AMA1 and RON2 proteins have diverged between Apicomplexa species, we show an intra-species conservation of their interaction. More importantly, invasion inhibition assays using recombinant proteins demonstrate that the RON2-AMA1 interaction is crucial for both T. gondii and P. falciparum entry into their host cells. This work provides the first evidence that AMA1 uses the rhoptry neck protein RON2 as a receptor to promote invasion by Apicomplexa parasites.

Structure-Function Relationship for a Divergent Atg8 Protein Required for a Nonautophagic Function in Apicomplexan Parasites.

Atg8 family proteins are highly conserved eukaryotic proteins with diverse autophagy and nonautophagic functions in eukaryotes. While the structural features required for conserved autophagy functions of Atg8 are well established, little is known about the molecular changes that facilitated acquisition of divergent, nonautophagic functions of Atg8. The malaria parasite Plasmodium falciparum offers a unique opportunity to study nonautophagic functions of Atg8 family proteins because it encodes a single Atg8 homolog whose only essential function is in the inheritance of an unusual secondary plastid called the apicoplast. Here, we used functional complementation to investigate the structure-function relationship for this divergent Atg8 protein. We showed that the LC3-interacting region (LIR) docking site (LDS), the major interaction interface of the Atg8 protein family, is required for P. falciparum Atg8 (PfAtg8) apicoplast localization and function, likely via Atg8 lipidation. On the other hand, another region previously implicated in canonical Atg8 interactions, the N-terminal helix, is not required for apicoplast-specific PfAtg8 function. Finally, our investigations at the cellular level demonstrate that the unique apicomplexan-specific loop, previously implicated in interaction with membrane conjugation machinery in recombinant protein-based in vitro assays, is not required for membrane conjugation nor for the apicoplast-specific effector function of Atg8 in both P. falciparum and related Apicomplexa member Toxoplasma gondii. These results suggest that the effector function of apicomplexan Atg8 is mediated by structural features distinct from those previously identified for macroautophagy and selective autophagy functions. IMPORTANCE The most extensively studied role of Atg8 proteins is in autophagy. However, it is clear that they have other nonautophagic functions critical to cell function and disease pathogenesis that are so far understudied compared to their canonical role in autophagy. Mammalian cells contain multiple Atg8 paralogs that have diverse, specialized functions. Gaining molecular insight into their nonautophagic functions is difficult because of redundancy between the homologs and their role in both autophagy and nonautophagic pathways. Malaria parasites such as Plasmodium falciparum are a unique system to study a novel, nonautophagic function of Atg8 separate from its role in autophagy: they have only one Atg8 protein whose only essential function is in the inheritance of the apicoplast, a unique secondary plastid organelle. Insights into the molecular basis of PfAtg8's function in apicoplast biogenesis will have important implications for the evolution of diverse nonautophagic functions of the Atg8 protein family.

Autophagy in protists and their hosts: When, how and why?

Pathogenic protists are a group of organisms responsible for causing a variety of human diseases including malaria, sleeping sickness, Chagas disease, leishmaniasis, and toxoplasmosis, among others. These diseases, which affect more than one billion people globally, mainly the poorest populations, are characterized by severe chronic stages and the lack of effective antiparasitic treatment. Parasitic protists display complex life-cycles and go through different cellular transformations in order to adapt to the different hosts they live in. Autophagy, a highly conserved cellular degradation process, has emerged as a key mechanism required for these differentiation processes, as well as other functions that are crucial to parasite fitness. In contrast to yeasts and mammals, protist autophagy is characterized by a modest number of conserved autophagy-related proteins (ATGs) that, even though, can drive the autophagosome formation and degradation. In addition, during their intracellular cycle, the interaction of these pathogens with the host autophagy system plays a crucial role resulting in a beneficial or harmful effect that is important for the outcome of the infection. In this review, we summarize the current state of knowledge on autophagy and other related mechanisms in pathogenic protists and their hosts. We sought to emphasize when, how, and why this process takes place, and the effects it may have on the parasitic cycle. A better understanding of the significance of autophagy for the protist life-cycle will potentially be helpful to design novel anti-parasitic strategies.

The moving junction of apicomplexan parasites: a key structure for invasion.

Most Apicomplexa are obligate intracellular parasites and many are important pathogens of human and domestic animals. For a successful cell invasion, they rely on their own motility and on a firm anchorage to their host cell, depending on the secretion of proteins and the establishment of a structure called the moving junction (MJ). The MJ moves from the apical to the posterior end of the parasite, leading to the internalization of the parasite into a parasitophorous vacuole. Based on recent data obtained in Plasmodium and Toxoplasma, an emerging model emphasizes a cooperative role of secreted parasitic proteins in building the MJ and driving this crucial invasive process. More precisely, the parasite exports the microneme protein AMA1 to its own surface and the rhoptry neck RON2 protein as a receptor inserted into the host cell together with other RON partners. Ongoing and future research will certainly help refining the model by characterizing the molecular organization within the MJ and its interactions with both host and parasite cytoskeleton for anchoring of the complex.

Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion.

One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ) between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1) and the neck of the rhoptries (for RON2/RON4/RON5 proteins), have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also demonstrated that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms in vitro. Finally, whereas AMA1 has previously been shown to be inserted into the parasite membrane upon secretion, we demonstrated, using differential permeabilization and loading of RON-specific antibodies into the host cell, that the RON complex is targeted to the host cell membrane, where RON4/5/8 remain associated with the cytoplasmic face. Globally, these results point toward a model of MJ organization where the parasite would be secreting and inserting interacting components on either side of the MJ, both at the host and at its own plasma membranes.

The pathogenicity and virulence of Toxoplasma gondii.

Toxoplasma gondii is a parasitic protist infecting a wide group of warm-blooded animals, ranging from birds to humans. While this infection is usually asymptomatic in healthy individuals, it can also lead to severe ocular or neurological outcomes in immunocompromised individuals or in developing fetuses. This obligate intracellular parasite has the ability to infect a considerable range of nucleated cells and can propagate in the intermediate host. Yet, under the pressure of the immune system it transforms into an encysted persistent form residing primarily in the brain and muscle tissues. Encysted parasites, which are resistant to current medication, may reactivate and give rise to an acute infection. The clinical outcome of toxoplasmosis depends on a complex balance between the host immune response and parasite virulence factors. Susceptibility to the disease is thus determined by both parasite strains and host species. Recent advances on our understanding of host cell-parasite interactions and parasite virulence have brought new insights into the pathophysiology of T. gondii infection and are summarized here.

The Autophagy Machinery in Human-Parasitic Protists; Diverse Functions for Universally Conserved Proteins.

Autophagy is a eukaryotic cellular machinery that is able to degrade large intracellular components, including organelles, and plays a pivotal role in cellular homeostasis. Target materials are enclosed by a double membrane vesicle called autophagosome, whose formation is coordinated by autophagy-related proteins (ATGs). Studies of yeast and Metazoa have identified approximately 40 ATGs. Genome projects for unicellular eukaryotes revealed that some ATGs are conserved in all eukaryotic supergroups but others have arisen or were lost during evolution in some specific lineages. In spite of an apparent reduction in the ATG molecular machinery found in parasitic protists, it has become clear that ATGs play an important role in stage differentiation or organelle maintenance, sometimes with an original function that is unrelated to canonical degradative autophagy. In this review, we aim to briefly summarize the current state of knowledge in parasitic protists, in the light of the latest important findings from more canonical model organisms. Determining the roles of ATGs and the diversity of their functions in various lineages is an important challenge for understanding the evolutionary background of autophagy.

Toxoplasma TgATG9 is critical for autophagy and long-term persistence in tissue cysts.

Many of the world's warm-blooded species are chronically infected with Toxoplasma gondii tissue cysts, including an estimated one-third of the global human population. The cellular processes that permit long-term persistence within the cyst are largely unknown for T. gondii and related coccidian parasites that impact human and animal health. Herein, we show that genetic ablation of TgATG9 substantially reduces canonical autophagy and compromises bradyzoite viability. Transmission electron microscopy revealed numerous structural abnormalities occurring in ∆atg9 bradyzoites. Intriguingly, abnormal mitochondrial networks were observed in TgATG9-deficient bradyzoites, some of which contained numerous different cytoplasmic components and organelles. ∆atg9 bradyzoite fitness was drastically compromised in vitro and in mice, with very few brain cysts identified in mice 5 weeks post-infection. Taken together, our data suggests that TgATG9, and by extension autophagy, is critical for cellular homeostasis in bradyzoites and is necessary for long-term persistence within the cyst of this coccidian parasite.

P18 (SRS35/TgSAG4) Plays a Role in the Invasion and Virulence of Toxoplasma gondii.

Toxoplasmosis is a prevalent parasitic disease caused by Toxoplasma gondii (T. gondii). Under the control of the host immune system, T. gondii persists as latent bradyzoite cysts. Immunosuppression leads to their reactivation, a potentially life-threatening condition. Interferon-gamma (IFN-γ) controls the different stages of toxoplasmosis. Here, we addressed the role of the parasite surface antigen P18, belonging to the Surface-Antigen 1 (SAG-1) Related Sequence (SRS) family, in a cyst-forming strain. Deletion of P18 gene (KO P18) impaired the invasion of parasites in macrophages and IFN-γ-mediated activation of macrophages further reduced the invasion capacity of this KO, as compared to WT strain. Mice infected by KO P18, showed a marked decrease in virulence during acute toxoplasmosis. This was consequent to less parasitemia, accompanied by a substantial recruitment of dendritic cells, macrophages and natural killer cells (NK). Furthermore, KO P18 resulted in a higher number of bradyzoite cysts, and a stronger inflammatory response. A prolonged survival of mice was observed upon immunosuppression of KO P18 infected BALB/c mice or upon oral infection of Severe Combined Immunodeficiency (SCID) mice, with intact macrophages and natural killer (NK) cells. In stark contrast, oral infection of NSG (NOD/Shi-scid/IL-2Rγnull) mice, defective in macrophages and NK cells, with KO P18, was as lethal as that of the control strain showing that the conversion from bradyzoites to tachyzoites is intact and, suggesting a role of P18 in the response to host IFN-γ. Collectively, these data demonstrate a role for P18 surface antigen in the invasion of macrophages and in the virulence of the parasite, during acute and chronic toxoplasmosis.