Identification of a Master Regulator of Differentiation in Toxoplasma.

Toxoplasma gondii chronically infects a quarter of the world's population, and its recrudescence can cause life-threatening disease in immunocompromised individuals and recurrent ocular lesions in the immunocompetent. Acute-stage tachyzoites differentiate into chronic-stage bradyzoites, which form intracellular cysts resistant to immune clearance and existing therapies. The molecular basis of this differentiation is unknown, despite being efficiently triggered by stresses in culture. Through Cas9-mediated screening and single-cell profiling, we identify a Myb-like transcription factor (BFD1) necessary for differentiation in cell culture and in mice. BFD1 accumulates during stress and its synthetic expression is sufficient to drive differentiation. Consistent with its function as a transcription factor, BFD1 binds the promoters of many stage-specific genes and represents a counterpoint to the ApiAP2 factors that dominate our current view of parasite gene regulation. BFD1 provides a genetic switch to study and control Toxoplasma differentiation and will inform prevention and treatment of chronic infections.

Invasion of Toxoplasma gondii bradyzoites: Molecular dissection of the moving junction proteins and effective vaccination targets.

Toxoplasmosis is a neglected parasitic disease necessitating public health control. Host cell invasion by Toxoplasma occurs at different stages of the parasite's life cycle and is crucial for survival and establishment of infection. In tachyzoites, which are responsible for acute toxoplasmosis, invasion involves the formation of a molecular bridge between the parasite and host cell membranes, referred to as the moving junction (MJ). The MJ is shaped by the assembly of AMA1 and RON2, as part of a complex involving additional RONs. While this essential process is well characterized in tachyzoites, the invasion process remains unexplored in bradyzoites, which form cysts and are responsible for chronic toxoplasmosis and contribute to the dissemination of the parasite between hosts. Here, we show that bradyzoites invade host cells in an MJ-dependent fashion but differ in protein composition from the tachyzoite MJ, relying instead on the paralogs AMA2 and AMA4. Functional characterization of AMA4 reveals its key role for cysts burden during the onset of chronic infection, while being dispensable for the acute phase. Immunizations with AMA1 and AMA4, alone or in complex with their rhoptry neck respective partners RON2 and RON2L1, showed that the AMA1-RON2 pair induces strong protection against acute and chronic infection, while the AMA4-RON2L1 complex targets more selectively the chronic form. Our study provides important insights into the molecular players of bradyzoite invasion and indicates that invasion of cyst-forming bradyzoites contributes to cyst burden. Furthermore, we validate AMA-RON complexes as potential vaccine candidates to protect against toxoplasmosis.

Iron Stress Affects the Growth and Differentiation of Toxoplasma gondii.

Iron is an indispensable nutrient for the survival of Toxoplasma gondii; however, excessive amounts can lead to toxicity. The parasite must overcome the host's "nutritional immunity" barrier and compete with the host for iron. Since T. gondii can infect most nucleated cells, it encounters increased iron stress during parasitism. This study assessed the impact of iron stress, encompassing both iron depletion and iron accumulation, on the growth of T. gondii. Iron accumulation disrupted the redox balance of T. gondii while enhancing the parasite's ability to adhere in high-iron environments. Conversely, iron depletion promoted the differentiation of tachyzoites into bradyzoites. Proteomic analysis further revealed proteins affected by iron depletion and identified the involvement of phosphotyrosyl phosphatase activator proteins in bradyzoite formation.

Factors Influencing Tissue Cyst Yield in a Murine Model of Chronic Toxoplasmosis.

Recent advances into the unique biology of Toxoplasma tissue cysts and the bradyzoites they house necessitate optimization of tissue cyst recovery from infected mouse brains. Here, we present data from 83 tissue cyst purifications of Type II ME49 tissue cysts in CBA/J mice performed over a period of 3 years. The effects of infection with both tissue culture tachyzoites as well as ex vivo tissue cysts were assessed. Significant mortality was restricted to tachyzoite infections with female mice being more susceptible. Infection with tissue cysts was associated with both lower overall symptomology and mortality, exhibiting no sex bias. Cumulatively, host sex did not impact overall tissue cyst yields, although tachyzoite-initiated infections generated significantly higher yields compared to tissue cyst-initiated infections. Notably, serial passage of tissue cysts was accompanied with a decreasing trend for subsequent cyst recovery. The time of tissue cyst harvest, a potential reflection of bradyzoite physiological state, had no significant impact on subsequent cyst yield at the selected time points. In aggregate, these data reveal the considerable heterogeneity associated with tissue cyst yield, making the design of adequately powered experiments critical. This is particularly the case for drug studies where overall tissue cyst burden is currently the primary and often sole metric of efficacy, as the data presented here demonstrate that cyst recovery between preparations of untreated animals can mirror and even exceed the reported effects of drug treatment.

Importance of aspartyl protease 5 in the establishment of the intracellular niche during acute and chronic infection of Toxoplasma gondii.

Virulence and persistence of the obligate intracellular parasite Toxoplasma gondii involve the secretion of effector proteins belonging to the family of dense granule proteins (GRAs) that act notably as modulators of the host defense mechanisms and participate in cyst wall formation. The subset of GRAs residing in the parasitophorous vacuole (PV) or exported into the host cell, undergo proteolytic cleavage in the Golgi upon the action of the aspartyl protease 5 (ASP5). In tachyzoites, ASP5 substrates play central roles in the morphology of the PV and the export of effectors across the translocon complex MYR1/2/3. Here, we used N-terminal amine isotopic labeling of substrates to identify novel ASP5 cleavage products by comparing the N-terminome of wild-type and Δasp5 lines in tachyzoites and bradyzoites. Validated substrates reside within the PV or PVM in an ASP5-dependent manner. Remarkably, Δasp5 bradyzoites are impaired in the formation of the cyst wall in vitro and exhibit a considerably reduced cyst burden in chronically infected animals. More specifically two-photon serial tomography of infected mouse brains revealed a comparatively reduced number and size of the cysts throughout the establishment of persistence in the absence of ASP5.

The Epigenome, Cell Cycle, and Development in Toxoplasma.

Toxoplasma gondii is a common veterinary and human pathogen that persists as latent bradyzoite forms within infected hosts. The ability of the parasite to interconvert between tachyzoite and bradyzoite is key for pathogenesis of toxoplasmosis, particularly in immunocompromised individuals. The transition between tachyzoites and bradyzoites is epigenetically regulated and coupled to the cell cycle. Recent epigenomic studies have begun to elucidate the chromatin states associated with developmental switches in T. gondii. Evidence is also emerging that AP2 transcription factors both activate and repress the bradyzoite developmental program. Further studies are needed to understand the mechanisms by which T. gondii transduces environmental signals to coordinate the epigenetic and transcriptional machinery that are responsible for tachyzoite-bradyzoite interconversion.

The beta subunit of AMP-activated protein kinase is critical for cell cycle progression and parasite development in Toxoplasma gondii.

Toxoplasma gondii is a widespread eukaryotic pathogen that causes life-threatening diseases in humans and diverse animals. It has a complex life cycle with multiple developmental stages, which are timely adjusted according to growth conditions. But the regulatory mechanisms are largely unknown. Here we show that the AMP-activated protein kinase (AMPK), a key regulator of energy homeostasis in eukaryotes, plays crucial roles in controlling the cell cycle progression and bradyzoite development in Toxoplasma. Deleting the ß regulatory subunit of AMPK in the type II strain ME49 caused massive DNA damage and increased spontaneous conversion to bradyzoites (parasites at chronic infection stage), leading to severe growth arrest and reduced virulence of the parasites. Under alkaline stress, all Δampkß mutants converted to a bradyzoite-like state but the cell division pattern was significantly impaired, resulting in compromised parasite viability. Moreover, we found that phosphorylation of the catalytic subunit AMPKα was greatly increased in alkaline stressed parasites, whereas AMPKß deletion mutants failed to do so. Phosphoproteomics found that many proteins with predicted roles in cell cycle and cell division regulation were differentially phosphorylated after AMPKß deletion, under both normal and alkaline stress conditions. Together, these results suggest that the parasite AMPK has critical roles in safeguarding cell cycle progression, and guiding the proper exist of the cell cycle to form mature bradyzoites when the parasites are stressed. Consistent with this model, growth of parasites was not significantly altered when AMPKß was deleted in a strain that was naturally reluctant to bradyzoite development.

4-Arylthiosemicarbazide Derivatives as Toxoplasmic Aromatic Amino Acid Hydroxylase Inhibitors and Anti-inflammatory Agents.

Approximately one-third of the human population is infected with the intracellular cosmopolitan protozoan Toxoplasma gondii (Tg), and a specific treatment for this parasite is still needed. Additionally, the increasing resistance of Tg to drugs has become a challenge for numerous research centers. The high selectivity of a compound toward the protozoan, along with low cytotoxicity toward the host cells, form the basis for further research, which aims at determining the molecular targets of the active compounds. Thiosemicarbazide derivatives are biologically active organic compounds. Previous studies on the initial preselection of 58 new 4-arylthiosemicarbazide derivatives in terms of their anti-Tg activity and selectivity made it possible to select two promising derivatives for further research. One of the important amino acids involved in the proliferation of Tg and the formation of parasitophorous vacuoles is tyrosine, which is converted by two unique aromatic amino acid hydroxylases to levodopa. Enzymatic studies with two derivatives (R: para-nitro and meta-iodo) and recombinant aromatic amino acid hydroxylase (AAHs) obtained in the E. coli expression system were performed, and the results indicated that toxoplasmic AAHs are a molecular target for 4-arylthiosemicarbazide derivatives. Moreover, the drug affinity responsive target stability assay also confirmed that the selected compounds bind to AAHs. Additionally, the anti-inflammatory activity of these derivatives was tested using THP1-Blue™ NF-κB reporter cells due to the similarity of the thiosemicarbazide scaffold to thiosemicarbazone, both of which are known NF-κB pathway inhibitors.

Toxoplasma gondii induces robust humoral immune response against cyst wall antigens in chronically infected animals and humans.

Toxoplasma gondii differentiation from proliferating tachyzoites into latent bradyzoites is central to pathogenesis and transmission. Strong humoral immune response has been reported against tachyzoite antigens, however, antibody-mediated response towards bradyzoite antigens is poorly characterized. This work aimed to study the humoral immune response towards bradyzoite and associated cyst wall antigens particularly CST1. The immunoreactivity of 404 goats, 88 sheep and 92 human sera to recombinant (CST1 and SRS9) and native proteins of encysted bradyzoite along with well-established tachyzoite antigens (SAG1 and GRA7) was determined using ELISA, Western blot and immunofluorescence analysis (IFA). ELISA results revealed nearly 50% of sera contain T. gondii specific antibodies. Results were further validated using Western blot and IFA. T. gondii positive sera predominantly recognized the cyst wall besides the known tachyzoite surface antigens. The presence of CST1 antibodies in seropositive samples were in line with the staining patterns which were consistent with CST localization. Notably, T. gondii IgM- IgG+ sera recognize the cyst wall whereas IgM + IgG-sera recognize tachyzoite antigens indicating acute infection consistent with presence of parasite DNA. The study demonstrates a strong humoral response against bradyzoite associated cyst wall antigens across naturally infected animals and humans. CST1 emerged as a key immunomodulatory antigen which may have direct implications for clinical immunodiagnostics.

In vitro activity of N-phenyl-1,10-phenanthroline-2-amines against tachyzoites and bradyzoites of Toxoplasma gondii.

Toxoplasma gondiiis an apicomplexan parasite, the causative agent of toxoplasmosis, a common disease in the world. Toxoplasmosis could be severe, especially in immunocompromised patients. The current therapy is limited, where pyrimethamine and sulfadiazine are the best choices despite being associated with side effects and ineffective against the bradyzoites, the parasitic form present during the chronic phase of the infection. Thus, new therapies against both tachyzoites and bradyzoites from T. gondii are urgent. Herein, we present the anti-T. gondii effect of 1,10-phenanthroline and its N-phenyl-1,10-phenanthroline-2-amine derivatives. The chemical modification of 1,10-phenanthroline tonew derivatives improved the anti-T. gondiiactivity 3.4 fold. The most active derivative presented ED50in the nanomolar range, the smallest value found was for Ph8, 0.1 µM for 96 h of treatment. The host cell viability was maintained after the treatment with the compounds, which were found to be highly selective presenting large selectivity indexes. Treatment with derivatives for 96 h was able to eliminate the T. gondii infection irreversibly. The ultrastructural alterations caused after the treatment with the most effective derivative (Ph8) included signs of cell death, specifically revealed by the Tunel assay for detection of DNA fragmentation. The Phen derivatives were also able to control the growth of the in vitro-derived bradyzoite forms of T. gondii EGS strain, causing its lysis and death. These findings promote the 1,10-phenanthroline derivatives as potential lead compounds for the development of a treatment for acute and chronic phases of toxoplasmosis.

Crossing of the Cystic Barriers of Toxoplasma gondii by the Fluorescent Coumarin Tetra-Cyclopeptide.

FR235222 is a natural tetra-cyclopeptide with a strong inhibition effect on histone deacetylases, effective on mammalian cells as well as on intracellular apicomplexan parasites, such as Toxoplasma gondii, in the tachyzoite and bradyzoite stages. This molecule is characterized by two parts: the zinc-binding group, responsible for the binding to the histone deacetylase, and the cyclic tetrapeptide moiety, which plays a crucial role in cell permeability. Recently, we have shown that the cyclic tetrapeptide coupled with a fluorescent diethyl-amino-coumarin was able to maintain properties of cellular penetration on human cells. Here, we show that this property can be extended to the crossing of the Toxoplasma gondii cystic cell wall and the cell membrane of the parasite in its bradyzoite form, while maintaining a high efficacy as a histone deacetylase inhibitor. The investigation by molecular modeling allows a better understanding of the penetration mechanism.

Toxoplasma gondii metacaspase 2 is an important factor that influences bradyzoite formation in the Pru strain.

Toxoplasma gondii is an important zoonotic protozoan of the phylum Apicomplexa that can infect nearly all warm-blooded animals. The parasite can exist as the interconvertible tachyzoite or bradyzoite forms, leading to acute or latent infection, respectively. No drug has been reported to penetrate the cyst wall and reduce bradyzoite survival and proliferation till now. The transcriptional level of metacaspases 2 (TgMCA2) in T. gondii is significantly upregulated during the formation of bradyzoites in the Pru strain, indicating that it may play an important role in the formation of bradyzoites. To further explore the function of TgMCA2, we constructed a TgMCA2 gene-knockout variant of the Pru strain (Δmca2). Comparative analysis revealed that the proliferative capacity of Pru Δmca2 increased, while the invasion and egressing properties were not affected by the knockout. Further data shows that the tachyzoites of Δmca2 failed to induce differentiation and form bradyzoites in vitro, and the transcriptional levels of some of the bradyzoite-specific genes (such as BAG1, LDH2, and SAG4A) in Δmca2 were significantly lower compared with that in the Pru strain at the bradyzoite stage. In vivo, no cysts were detected in Δmca2-infected mice. Further determination of parasite burden in Δmca2- and Pru-infected mice brain tissue at the genetic level showed that the gene load was significantly lower than that in Pru. In summary, we confirmed that TgMCA2 contributes to the formation of bradyzoites, and could provide an important foundation for the development of attenuated vaccines for the prevention of T. gondii infection.

Screening of compound libraries for inhibitors of Toxoplasma growth and invasion.

Toxoplasma gondii can infect virtually all warm-blooded animals, including humans. It can differentiate between rapidly replicating tachyzoites that cause acute infection and slowly growing bradyzoites in tissue cysts. Treatment options for toxoplasmosis are challenging because current therapies cannot eradicate the latent T. gondii infection that is mainly caused by the bradyzoite forms. Accordingly, recurrence of infection is a problem for immunocompromised patients and congenitally infected patients. Protein kinases have been widely studied in eukaryotic cells, and while little is known about signaling in Toxoplasma infection, it is likely that protein kinases play a key role in parasite proliferation, differentiation, and probably invasion. To identify optimized new kinase inhibitors for drug development against T. gondii, we screened a library of kinase inhibitor compounds for anti-Toxoplasma activity and host cell cytotoxicity. Pyrimethamine served as a positive control and 0.5% DMSO was used as a negative control. Among the 80 compounds screened, 6 compounds demonstrated ≥ 80% parasite growth inhibition at concentrations at which 5 compounds did not suppress host cell viability, while 3 kinase inhibitors (Bay 11-7082, Tyrphostin AG 1295 and PD-98059) had suppressive effects individually on parasite growth and host cell invasion, but did not strongly induce bradyzoite formation.

Downregulation of the Central Noradrenergic System by Toxoplasma gondii Infection.

Toxoplasma gondii is associated with physiological effects in the host. Dysregulation of catecholamines in the central nervous system has previously been observed in chronically infected animals. In the study described here, the noradrenergic system was found to be suppressed with decreased levels of norepinephrine (NE) in brains of infected animals and in infected human and rat neural cells in vitro The mechanism responsible for the NE suppression was found to be downregulation of dopamine ß-hydroxylase (DBH) gene expression, encoding the enzyme that synthesizes norepinephrine from dopamine, with downregulation observed in vitro and in infected brain tissue, particularly in the dorsal locus coeruleus/pons region. The downregulation was sex specific, with males expressing reduced DBH mRNA levels whereas females were unchanged. Rather, DBH expression correlated with estrogen receptor in the female rat brains for this estrogen-regulated gene. DBH silencing was not a general response of neurons to infection, as human cytomegalovirus did not downregulate DBH expression. The noradrenergic-linked behaviors of sociability and arousal were altered in chronically infected animals, with a high correlation between DBH expression and infection intensity. A decrease in DBH expression in noradrenergic neurons can elevate dopamine levels, which provides a possible explanation for mixed observations of changes in this neurotransmitter with infection. Decreased NE is consistent with the loss of coordination and motor impairments associated with toxoplasmosis. Further, the altered norepinephrine synthesis observed here may, in part, explain behavioral effects of infection and associations with mental illness.

A latent ability to persist: differentiation in Toxoplasma gondii.

A critical factor in the transmission and pathogenesis of Toxoplasma gondii is the ability to convert from an acute disease-causing, proliferative stage (tachyzoite), to a chronic, dormant stage (bradyzoite). The conversion of the tachyzoite-containing parasitophorous vacuole membrane into the less permeable bradyzoite cyst wall allows the parasite to persist for years within the host to maximize transmissibility to both primary (felids) and secondary (virtually all other warm-blooded vertebrates) hosts. This review presents our current understanding of the latent stage, including the factors that are important in bradyzoite induction and maintenance. Also discussed are the recent studies that have begun to unravel the mechanisms behind stage switching.

Micronemal protein 13 contributes to the optimal growth of Toxoplasma gondii under stress conditions.

Toxoplasma gondii is a ubiquitous parasitic protozoan infecting humans and a wide variety of animals. Fast-replicating tachyzoites during acute infection and slowly growing bradyzoites during chronic infection are the two basic forms of T. gondii in intermediate hosts. Interconversion between the two contributes to the transmission and pathogenesis of this parasite. Secretory micronemal proteins are thought to mediate interactions with host cells and facilitate parasite invasion, therefore the majority of them are highly expressed in tachyzoites. Micronemal protein 13 (MIC13) is unique in that its expression is low in tachyzoites and is upregulated under bradyzoite-inducing conditions. Previous attempts to disrupt this gene were not successful, implying that it may play critical roles during parasite growth. However, in this study, MIC13 was successfully disrupted in type 1 strain RH and type 2 strain ME49 using CRISPR/Cas9-mediated gene disruption techniques. Consistent with its low expression in tachyzoites and increased expression under stress or bradyzoite-inducing conditions, MIC13-inactivated mutants displayed normal growth, host cell invasion, intracellular replication, and egress, as well as acute virulence at the tachyzoite stage. However, under stress conditions, such as high pH or oxygen limitation, MIC13-disrupted parasites showed significantly slower growth rates compared to the parental strains, suggesting that it is required for optimal parasite growth under bradyzoite-inducing or stress conditions. This is the first micronemal protein reported to have such expression pattern and function modes, which expands our understanding of the diverse functions of micronemal proteins.

Apicoplast fatty acid synthesis is essential for pellicle formation at the end of cytokinesis in Toxoplasma gondii.

The apicomplexan protozoan Toxoplasma gondii, the causative agent of toxoplasmosis, harbors an apicoplast, a plastid-like organelle with essential metabolic functions. Although the FASII fatty acid biosynthesis pathway located in the apicoplast is essential for parasite survival, the cellular effects of FASII disruption in T. gondii had not been examined in detail. Here, we combined light and electron microscopy techniques - including focused ion beam scanning electron microscopy (FIB-SEM) - to characterize the effect of FASII disruption in T. gondii, by treatment with the FASII inhibitor triclosan or by inducible knockdown of the FASII component acyl carrier protein. Morphological analyses showed that FASII disruption prevented cytokinesis completion in T. gondii tachyzoites, leading to the formation of large masses of 'tethered' daughter cells. FIB-SEM showed that tethered daughters had a mature basal complex, but a defect in new membrane addition between daughters resulted in incomplete pellicle formation. Addition of exogenous fatty acids to medium suppressed the formation of tethered daughter cells and supports the notion that FASII is essential to generate lipid substrates required for the final step of parasite division.

Toxoplasma gondii reorganizes the host cell architecture during spontaneous cyst formation in vitro.

Toxoplasma gondii is an intracellular protozoan parasite that causes toxoplasmosis, a prevalent infection related to abortion, ocular diseases and encephalitis in immuno-compromised individuals. In the untreatable (and life-long) chronic stage of toxoplasmosis, parasitophorous vacuoles (PVs, containing T. gondii tachyzoites) transform into tissue cysts, containing slow-dividing bradyzoite forms. While acute-stage infection with tachyzoites involves global rearrangement of the host cell cytoplasm, focused on favouring tachyzoite replication, the cytoplasmic architecture of cells infected with cysts had not been described. Here, we characterized (by fluorescence and electron microscopy) the redistribution of host cell structures around T. gondii cysts, using a T. gondii strain (EGS) with high rates of spontaneous cystogenesis in vitro. Microtubules and intermediate filaments (but not actin microfilaments) formed a 'cage' around the cyst, and treatment with taxol (to inhibit microtubule dynamics) favoured cystogenesis. Mitochondria, which appeared adhered to the PV membrane, were less closely associated with the cyst wall. Endoplasmic reticulum (ER) profiles were intimately associated with folds in the cyst wall membrane. However, the Golgi complex was not preferentially localized relative to the cyst, and treatment with tunicamycin or brefeldin A (to disrupt Golgi or ER function, respectively) had no significant effect on cystogenesis. Lysosomes accumulated around cysts, while early and late endosomes were more evenly distributed in the cytoplasm. The endocytosis tracer HRP (but not BSA or transferrin) reached bradyzoites after uptake by infected host cells. These results suggest that T. gondii cysts reorganize the host cell cytoplasm, which may fulfil specific requirements of the chronic stage of infection.

The antifungal Aureobasidin A and an analogue are active against the protozoan parasite Toxoplasma gondii but do not inhibit sphingolipid biosynthesis.

Toxoplasma gondii is an obligate intracellular protozoan parasite of the phylum Apicomplexa, and toxoplasmosis is an important disease of both humans and economically important animals. With a limited array of drugs available there is a need to identify new therapeutic compounds. Aureobasidin A (AbA) is an antifungal that targets the essential inositol phosphorylceramide (IPC, sphingolipid) synthase in pathogenic fungi. This natural cyclic depsipeptide also inhibits Toxoplasma proliforation, with the protozoan IPC synthase orthologue proposed as the target. The data presented here show that neither AbA nor an analogue (Compound 20), target the protozoan IPC synthase orthologue or total parasite sphingolipid synthesis. However, further analyses confirm that AbA exhibits significant activity against the proliferative tachyzoite form of Toxoplasma, and Compound 20, whilst effective, has reduced efficacy. This difference was more evident on analyses of the direct effect of these compounds against isolated Toxoplasma, indicating that AbA is rapidly microbicidal. Importantly, the possibility of targeting the encysted, bradyzoite, form of the parasite with AbA and Compound 20 was demonstrated, indicating that this class of compounds may provide the basis for the first effective treatment for chronic toxoplasmosis.

Induction of Autophagy interferes the tachyzoite to bradyzoite transformation of Toxoplasma gondii.

Autophagy process in Toxoplasma gondii plays a vital role in regulating parasite survival or death. Thus, once having an understanding of certain effects of autophagy on the transformation of tachyzoite to bradyzoite this will allow us to elucidate the function of autophagy during parasite development. Herein, we used three TgAtg proteins involved in Atg8 conjugation system, TgAtg3, TgAtg7 and TgAtg8 to evaluate the autophagy level in tachyzoite and bradyzoite of Toxoplasma in vitro based on Pru TgAtg7-HA transgenic strains. We showed that both TgAtg3 and TgAtg8 were expressed at a significantly lower level in bradyzoites than in tachyzoites. Importantly, the number of parasites containing fluorescence-labelled TgAtg8 puncta was significantly reduced in bradyzoites than in tachyzoites, suggesting that autophagy is downregulated in Toxoplasma bradyzoite in vitro. Moreover, after treatment with drugs, bradyzoite-specific gene BAG1 levels decreased significantly in rapamycin-treated bradyzoites and increased significantly in 3-MA-treated bradyzoites in comparison with control bradyzoites, indicating that Toxoplasma autophagy is involved in the transformation of tachyzoite to bradyzoite in vitro. Together, it is suggested that autophagy may serve as a potential strategy to regulate the transformation.