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.

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 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.

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.

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.

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.

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.

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.

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.

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.

The Bradyzoite: A Key Developmental Stage for the Persistence and Pathogenesis of Toxoplasmosis.

Toxoplasma gondii is a ubiquitous parasitic protist found in a wide variety of hosts, including a large proportion of the human population. Beyond an acute phase which is generally self-limited in immunocompetent individuals, the ability of the parasite to persist as a dormant stage, called bradyzoite, is an important aspect of toxoplasmosis. Not only is this stage not eliminated by current treatments, but it can also reactivate in immunocompromised hosts, leading to a potentially fatal outcome. Yet, despite its critical role in the pathology, the bradyzoite stage is relatively understudied. One main explanation is that it is a considerably challenging model, which essentially has to be derived from in vivo sources. However, recent progress on genetic manipulation and in vitro differentiation models now offers interesting perspectives for tackling key biological questions related to this particularly important developmental stage.

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.

The role of host autophagy machinery in controlling Toxoplasma infection.

Toxoplasma gondii is an obligate intracellular parasitic protist that infects a wide range of warm-blooded vertebrates. Although this parasite can cause serious complications, infections are often asymptomatic, allowing T. gondii to persist in its host and possibly enhancing the chances of its transmission. T. gondii has thus evolved multiple mechanisms of host manipulation to establish chronic infection. This persistence involves a balance between host immunity and parasite evasion of this immune response. This review highlights recent investigations that have demonstrated the important role played by the autophagy machinery in this balance, both in parasite control by the host, and in host exploitation by the parasite.

Characterisation of two Toxoplasma PROPPINs homologous to Atg18/WIPI suggests they have evolved distinct specialised functions.

Toxoplasma gondii is a parasitic protist possessing a limited set of proteins involved in the autophagy pathway, a self-degradative machinery for protein and organelle recycling. This distant eukaryote has even repurposed part of this machinery, centered on protein ATG8, for a non-degradative function related to the maintenance of the apicoplast, a parasite-specific organelle. However, some evidence also suggest Toxoplasma is able to generate autophagic vesicles upon stress, and that some autophagy-related proteins, such as ATG9, might be involved solely in the canonical autophagy function. Here, we have characterised TgPROP1 and TgPROP2, two Toxoplasma proteins containing WD-40 repeat that can bind lipids for their recruitment to vesicular structures upon stress. They belong to the PROPPIN family and are homologues to ATG18/WIPI, which are known to be important for the autophagic process. We conducted a functional analysis of these two Toxoplasma PROPPINs. One of them is dispensable for normal in vitro growth, although it may play a role for parasite survival in specific stress conditions or for parasite fitness in the host, through a canonical autophagy-related function. The other, however, seems important for parasite viability in normal growth conditions and could be primarily involved in a non-canonical function. These divergent roles for two proteins from the same family illustrate the functional versatility of the autophagy-related machinery in Toxoplasma.

RON4L1 is a new member of the moving junction complex in Toxoplasma gondii.

Apicomplexa parasites, including Toxoplasma and Plasmodium species, possess a unique invasion mechanism that involves a tight apposition between the parasite and the host plasma membranes, called "moving junction" (MJ). The MJ is formed by the assembly of the microneme protein AMA1, exposed at the surface of the parasite, and the parasite rhoptry neck (RON) protein RON2, exposed at the surface of the host cell. In the host cell, RON2 is associated with three additional parasite RON proteins, RON4, RON5 and RON8. Here we describe RON4L1, an additional member of the MJ complex in Toxoplasma. RON4L1 displays some sequence similarity with RON4 and is cleaved at the C-terminal end before reaching the rhoptry neck. Upon secretion during invasion, RON4L1 is associated with MJ and targeted to the cytosolic face of the host membrane. We generated a RON4 L1 knock-out cell line and showed that it is not essential for the lytic cycle in vitro, although mutant parasites kill mice less efficiently. Similarly to RON8, RON4L1 is a coccidian-specific protein and its traffic to the MJ is not affected in absence of RON2, RON4 and RON5, suggesting the co-existence of independent MJ complexes in tachyzoite of Toxoplasma.

Autophagy in apicomplexan parasites.

Autophagy is a highly conserved eukaryotic degradation process that permits the recycling of intracellular components. The molecular machinery and the functions of autophagy have been classically characterized in mammalian cells and yeast, but have long remained unexplored in less-studied eukaryotes. Apicomplexan parasites are early-diverging eukaryotes responsible for a number of important human and veterinary diseases. In light of recent investigations into autophagy function in two of these pathogens, Plasmodium and Toxoplasma, it seems their autophagy-related machinery could be involved in both a canonical degradative function, and a non-canonical role related to the apicoplast, a metabolically important organelle of endosymbiotic origin.

Autophagy participates in the unfolded protein response in Toxoplasma gondii.

Environmental and genetic perturbations of endoplasmic reticulum (ER) function can lead to the accumulation of unfolded proteins. In these conditions, eukaryotic cells can activate a complex signaling network called the unfolded protein response (UPR) to reduce ER stress and restore cellular homeostasis. Autophagy, a degradation and recycling process, is part of this response. The parasitic protist Toxoplasma gondii is known to be able to activate the UPR upon ER stress, and we now show that this pathway leads to autophagy activation, supporting the idea of a regulated function for canonical autophagy as part of an integrated stress response in the parasites.

Toxoplasma depends on lysosomal consumption of autophagosomes for persistent infection.

Globally, nearly 2 billion people are infected with the intracellular protozoan Toxoplasma gondii1. This persistent infection can cause severe disease in immunocompromised people and is epidemiologically linked to major mental illnesses2 and cognitive impairment3. There are currently no options for curing this infection. The lack of effective therapeutics is due partly to a poor understanding of the essential pathways that maintain long-term infection. Although it is known that Toxoplasma replicates slowly within intracellular cysts demarcated with a cyst wall, precisely how it sustains itself and remodels organelles in this niche is unknown. Here, we identify a key role for proteolysis within the parasite lysosomal organelle (the vacuolar compartment or VAC) in turnover of autophagosomes and persistence during neural infection. We found that disrupting a VAC-localized cysteine protease compromised VAC digestive function and markedly reduced chronic infection. Death of parasites lacking the VAC protease was preceded by accumulation of undigested autophagosomes in the parasite cytoplasm. These findings suggest an unanticipated function for parasite lysosomal degradation in chronic infection, and identify an intrinsic role for autophagy in the T. gondii parasite and its close relatives. This work also identifies a key element of Toxoplasma persistence and suggests that VAC proteolysis is a prospective target for pharmacological development.

Apicomplexan autophagy and modulation of autophagy in parasite-infected host cells.

Apicomplexan parasites are responsible for a number of important human pathologies. Obviously, as Eukaryotes they share a number of cellular features and pathways with their respective host cells. One of them is autophagy, a process involved in the degradation of the cell's own components. These intracellular parasites nonetheless seem to present a number of original features compared to their very evolutionarily distant host cells. In mammals and other metazoans, autophagy has been identified as an important contributor to the defence against microbial pathogens. Thus, host autophagy also likely plays a key role in the control of apicomplexan parasites, although its potential manipulation and subversion by intracellular parasites creates a complex interplay in the regulation of host and parasite autophagy. In this mini-review, we summarise current knowledge on autophagy in both parasites and their host cells, in the context of infection by three Apicomplexa: Plasmodium, Toxoplasma, and Theileria.