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BACKGROUND: Chicken coccidiosis is an intracellular parasitic disease that presents major challenges to the development of the commercial poultry industry. Perennial drug selective pressure has led to the multi-drug resistance of chicken coccidia, which makes the prevention and control of chicken coccidiosis extremely difficult. In recent years, natural plant products have attracted the attention of researchers due to their inherent advantages, such as the absence of veterinary drug residues. The development of these natural products provides a new direction for the prevention and treatment of chicken coccidiosis. METHODS: The anticoccidial effect of a natural plant product combination formulation (eucalyptus oil + apigenin + eugenol essential oil) was tested against Eimeria tenella in broilers. To search for the optimal concentration of the combination formulation, we screened 120 broilers in a chicken cage trial in which 100 broilers were infected with 5 × 104 sporulated Eimeria tenella oocysts; broilers receiving a decoquinate solution was set up as a chemical control. The optimal anticoccidial concentration was determined by calculating the anticoccidial index (ACI), and the suitable concentration was used as the recommended dose for a series of safety dose assessment tests, such as feed conversion ratio (FCR), hematological indices and serum biochemical indices, as well as liver and kidney sections, at onefold (low dose), threefold (medium dose) and sixfold (high dose) the recommended dose (RD). RESULTS: The results showed that this combination formulation of three plant natural products had a better anticoccidial effect than formulations containing two plant natural products or a single one, with an ACI of 169.3. The dose gradient anticoccidial test revealed that the high-dose formulation group had a better anticoccidial effect (ACI = 169.2) than the medium- and low-dose groups. The safety evaluation test showed that concentrations of the formulation at one-, three- and sixfold the RD were non-toxic to Arbor Acres broilers, indicating the high safety of the combination formulation. CONCLUSIONS: The combination formulation showed not only a moderate anticoccidial effect but also had a high safety profile for broilers. The results of this study indicate a new alternative for the prevention and control of coccidiosis in broilers.
Assuntos
Galinhas , Coccidiose , Coccidiostáticos , Eimeria tenella , Eucalyptus , Eugenol , Doenças das Aves Domésticas , Animais , Galinhas/parasitologia , Eimeria tenella/efeitos dos fármacos , Coccidiose/tratamento farmacológico , Coccidiose/veterinária , Coccidiose/parasitologia , Doenças das Aves Domésticas/tratamento farmacológico , Doenças das Aves Domésticas/parasitologia , Coccidiostáticos/farmacologia , Coccidiostáticos/uso terapêutico , Coccidiostáticos/administração & dosagem , Eugenol/farmacologia , Eugenol/administração & dosagem , Eucalyptus/química , Produtos Biológicos/farmacologia , Produtos Biológicos/administração & dosagem , Oocistos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Extratos Vegetais/administração & dosagem , Óleos Voláteis/farmacologia , Óleos Voláteis/química , Óleos Voláteis/administração & dosagemRESUMO
Chicken coccidiosis causes retarded growth and low production performance in poultry, resulting in huge economic losses to the poultry industry. In order to prevent and control chicken coccidiosis, great efforts have been made to develop new drugs and vaccines, which require pure isolates of Eimeria spp. In this study, we obtained the Eimeira tenella Xiantao isolate by single oocyst isolation technology and compared its genome with the reference genome GCF_000499545.2_ETH001 of the Houghton strain. The results of the comparative genomic analysis indicated that the genome of this isolate contained 46,888 single nucleotide polymorphisms (SNPs). There were 15,107 small insertion and deletion variations (indels), 1693 structural variations (SV), and 3578 copy number variations (CNV). In addition, 64 broilers were used to determine the resistance profile of Xiantao strain. Drug susceptibility testing revealed that this isolate was completely resistant to monensin, diclazuril, halofuginone, sulfachlorpyrazine sodium, and toltrazuril, but sensitive to decoquinate. These data improve our understanding of drug resistance in avian coccidia.
Assuntos
Galinhas , Coccidiose , Resistência a Medicamentos , Eimeria tenella , Doenças das Aves Domésticas , Eimeria tenella/genética , Eimeria tenella/efeitos dos fármacos , Eimeria tenella/isolamento & purificação , Animais , China , Galinhas/parasitologia , Doenças das Aves Domésticas/parasitologia , Coccidiose/veterinária , Coccidiose/parasitologia , Resistência a Medicamentos/genética , Coccidiostáticos/farmacologia , Polimorfismo de Nucleotídeo Único , Genoma de ProtozoárioRESUMO
Toxoplasma gondii (T. gondii)-associated polymorphic effector proteins are crucial in parasite development and regulating host anti-T. gondii immune responses. However, the mechanism remains obscure. Here, it is shown that Toxoplasma effector dense granules 4 (GRA4) restricts host IFN-I activation. Infection with Δgra4 mutant T. gondii strain induces stronger IFN-I responses and poses a severe threat to host health. Mechanistically, GRA4 binds to phosphorylated TBK1 to promote TRIM27-catalyzed K48-ubiquitination at Lys251/Lys372 residues, which enhances its recognition by autophagy receptor p62, ultimately leading to TBK1 autophagic degradation. Furthermore, an avirulent Δgra4 strain (ME49Δompdc/gra4) is constructed for tumor immunotherapy due to its ability to enhance IFN-I production. Earlier vaccination with ME49Δompdc/gra4 confers complete host resistance to the tumor compared with the classical ME49Δompdc treatment. Notably, ME49Δompdc/gra4 vaccination induces a specific CD64+MAR-1+CD11b+ dendritic cell subset, thereby enhancing T cell anti-tumor responses. Overall, these findings identify the negative role of T. gondii GRA4 in modulating host IFN-I signaling and suggest that GRA4 can be a potential target for the development of T. gondii vaccines and tumor immunotherapy.
Assuntos
Imunoterapia , Proteínas Serina-Treonina Quinases , Proteínas de Protozoários , Toxoplasma , Toxoplasma/imunologia , Toxoplasma/metabolismo , Toxoplasma/genética , Imunoterapia/métodos , Animais , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Camundongos Endogâmicos C57BL , Toxoplasmose/imunologia , Toxoplasmose/metabolismo , Toxoplasmose/genética , Modelos Animais de Doenças , Humanos , Neoplasias/imunologia , Neoplasias/terapia , Neoplasias/metabolismoRESUMO
Citrate synthase catalyzes the first and the rate-limiting reaction of the tricarboxylic acid (TCA) cycle, producing citrate from the condensation of oxaloacetate and acetyl-coenzyme A. The parasitic protozoan Toxoplasma gondii has full TCA cycle activity, but its physiological roles remain poorly understood. In this study, we identified three proteins with predicted citrate synthase (CS) activities two of which were localized in the mitochondrion, including the 2-methylcitrate synthase (PrpC) that was thought to be involved in the 2-methylcitrate cycle, an alternative pathway for propionyl-CoA detoxification. Further analyses of the two mitochondrial enzymes showed that both had citrate synthase activity, but the catalytic efficiency of CS1 was much higher than that of PrpC. Consistently, the deletion of CS1 resulted in a significantly reduced flux of glucose-derived carbons into TCA cycle intermediates, leading to decreased parasite growth. In contrast, disruption of PrpC had little effect. On the other hand, simultaneous disruption of both CS1 and PrpC resulted in more severe metabolic changes and growth defects than a single deletion of either gene, suggesting that PrpC does contribute to citrate production under physiological conditions. Interestingly, deleting Δcs1 and Δprpc individually or in combination only mildly or negligibly affected the virulence of parasites in mice, suggesting that both enzymes are dispensable in vivo. The dispensability of CS1 and PrpC suggests that either the TCA cycle is not essential for the asexual reproduction of tachyzoites or there are other routes of citrate supply in the parasite mitochondrion.
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Pyruvate lies at a pivotal node of carbon metabolism in eukaryotes. It is involved in diverse metabolic pathways in multiple organelles, and its interorganelle shuttling is crucial for cell fitness. Many apicomplexan parasites harbor a unique organelle called the apicoplast that houses metabolic pathways like fatty acid and isoprenoid precursor biosyntheses, requiring pyruvate as a substrate. However, how pyruvate is supplied in the apicoplast remains enigmatic. Here, deploying the zoonotic parasite Toxoplasma gondii as a model apicomplexan, we identified two proteins residing in the apicoplast membranes that together constitute a functional apicoplast pyruvate carrier (APC) to mediate the import of cytosolic pyruvate. Depletion of APC results in reduced activities of metabolic pathways in the apicoplast and impaired integrity of this organelle, leading to parasite growth arrest. APC is a pyruvate transporter in diverse apicomplexan parasites, suggesting a common strategy for pyruvate acquisition by the apicoplast in these clinically relevant intracellular pathogens.
Assuntos
Apicoplastos , Ácido Pirúvico , Toxoplasma , Apicoplastos/metabolismo , Toxoplasma/metabolismo , Ácido Pirúvico/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Animais , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Transporte Biológico , Redes e Vias MetabólicasRESUMO
Toxoplasma gondii is a zoonotic parasite infecting humans and nearly all warm-blooded animals. Successful parasitism in diverse hosts at various developmental stages requires the parasites to fine tune their metabolism according to environmental cues and the parasite's needs. By manipulating the ß and γ subunits, we have previously shown that AMP-activated protein kinase (AMPK) has critical roles in regulating the metabolic and developmental programmes. However, the biological functions of the α catalytic subunit have not been established. T. gondii encodes a canonical AMPKα, as well as a KIN kinase whose kinase domain has high sequence similarities to those of classic AMPKα proteins. Here, we found that TgKIN is dispensable for tachyzoite growth, whereas TgAMPKα is essential. Depletion of TgAMPKα expression resulted in decreased ATP levels and reduced metabolic flux in glycolysis and the tricarboxylic acid cycle, confirming that TgAMPK is involved in metabolic regulation and energy homeostasis in the parasite. Sequential truncations at the C-terminus found an α-helix that is key for the function of TgAMPKα. The amino acid sequences of this α-helix are not conserved among various AMPKα proteins, likely because it is involved in interactions with TgAMPKß, which only have limited sequence similarities to AMPKß in other eukaryotes. The essential role of the less conserved C-terminus of TgAMPKα provides opportunities for parasite specific drug designs targeting TgAMPKα.
Assuntos
Parasitos , Toxoplasma , Animais , Humanos , Proteínas Quinases Ativadas por AMP , Sequência de Aminoácidos , Proliferação de CélulasRESUMO
IMPORTANCE: Sexual development is vital for the transmission, genetic hybridization, and population evolution of apicomplexan pathogens, which include several clinically relevant parasites, such as Plasmodium, Eimeria, and Toxoplasma gondii. Previous studies have demonstrated different morphological characteristics and division patterns between asexual and sexual stages of the parasites. However, the primary regulation is poorly understood. A transition from the asexual to the sexual stage is supposedly triggered/accompanied by rewiring of gene expression and controlled by transcription factors and chromatin modulators. Herein, we discovered a tachyzoite-specific transcriptional factor AP2XII-1, which represses the presexual development in the asexual tachyzoite stage of T. gondii. Conditional knockdown of AP2XII-1 perturbs tachyzoite proliferation by endodyogeny and drives a transition to a morphologically and transcriptionally distinct merozoite stage. The results also suggest a hierarchical transcriptional regulation of sexual development by AP2 factors and provide a path to culturing merozoites and controlling inter-host transmission of T. gondii.
Assuntos
Toxoplasma , Animais , Toxoplasma/metabolismo , Fatores de Transcrição/metabolismo , Cromatina/metabolismo , Regulação da Expressão Gênica , Merozoítos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
Toxoplasma gondii is a ubiquitous pathogen that infects all warm-blooded animals, including humans, causing substantial socioeconomic and healthcare burdens. However, there is no ideal vaccine for toxoplasmosis. As metabolism is important in the growth and virulence of Toxoplasma, some key pathways are promising antiparasitic targets. Here, we identified 6-phosphogluconate dehydrogenase 1 (Tg6PGDH1) in the oxidative pentose phosphate pathway as a cytoplasmic protein that is dispensable for tachyzoite growth of T. gondii in vitro but critical for virulence and cyst formation in vivo. The depletion of Tg6PGDH1 causes decreased gene transcription involved in signal transduction, transcriptional regulation and virulence. Furthermore, we analysed the protective effect of the ME49Δ6pgdh1 mutant as an attenuated vaccine and found that ME49Δ6pgdh1 immunization stimulated strong protective immunity against lethal challenges and blocked cyst formation caused by reinfection. Furthermore, we showed that ME49Δ6pgdh1 immunization stimulated increased levels of interferon-gamma, tumour necrosis factor-alpha and Toxoplasma-specific IgG antibodies. These data highlight the role of Tg6PGDH1 in the growth and virulence of T. gondii and its potential as a target for the development of a live-attenuated vaccine.
RESUMO
Metabolism associated with energy production is highly compartmentalized in eukaryotic cells. During this process, transporters that move metabolites across organelle membranes play pivotal roles. The highly conserved ADP/ATP carrier (AAC) involved in ATP and ADP exchange between the mitochondria and cytoplasm is key to linking the metabolic activities in these 2 compartments. The ATP produced in mitochondria can be exchanged with cytoplasmic ADP by AAC, thus satisfying the energy needs in the cytoplasm. Toxoplasma gondii is an obligate intracellular parasite with a wide range of hosts. Previous studies have shown that mitochondrial metabolism helps Toxoplasma to parasitize diverse host cells. Here, we identified 2 putative mitochondria ADP/ATP carriers in Toxoplasma with significant sequence similarity to known AACs from other eukaryotes. We examined the ATP transport function of TgAACs by expressing them in Escherichia coli cells and found that only TgAAC1 had ATP transport activity. Moreover, knockdown of TgAAC1 caused severe growth defects of parasites and heterologous expression of mouse ANT2 in the TgAAC1 depletion mutant restored its growth, revealing its importance for parasite growth. These results verified that TgAAC1 functions as the mitochondrial ADP/ATP carrier in T. gondii and the functional studies demonstrated the importance of TgAAC1 for tachyzoites growth. IMPORTANCE T. gondii has an efficient and flexible energy metabolism system to meet different growth needs. ATP is an energy-carrying molecule and needs to be exchanged between organelles with the assistance of transporters. However, the function of TgAACs has yet to be characterized. Here, we identified 2 putative AACs of T. gondii and verified that only TgAAC1 had ATP transport activity with expression in the intact E. coli cells. Detailed analyses found that TgAAC1 is critical for the growth of tachyzoites and TgAAC2 is dispensable. Moreover, complementation with mouse ANT2 restored the growth speed of iTgAAC1, further suggesting TgAAC1 functions as a mitochondrial ADP/ATP carrier. Our research demonstrated the importance of TgAAC1 for tachyzoites growth.
Assuntos
Parasitos , Toxoplasma , Animais , Camundongos , Parasitos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
Toxoplasma gondii is an obligate intracellular parasite capable of infecting humans and animals. The organism has extraordinary metabolic resilience that allows it to establish parasitism in varied nutritional milieus of diverse host cells. Our earlier work has shown that, despite flexibility in the usage of glucose and glutamine as the major carbon precursors, the production of pyruvate by glycolytic enzymes is central to the parasite's growth. Pyruvate is metabolized in a number of subcellular compartments, including the mitochondrion, apicoplast, and cytosol. With the objective of examining the mechanism and importance of the mitochondrial pool of pyruvate imported from the cytosol, we identified the conserved mitochondrial pyruvate carrier (MPC) complex, consisting of two subunits, MPC1 and MPC2, in T. gondii. The two parasite proteins could complement a yeast mutant deficient in growth on leucine and valine. Genetic ablation of either one or both subunits reduced the parasite's growth, mimicking the deletion of branched-chain ketoacid dehydrogenase (BCKDH), which has been reported to convert pyruvate into acetyl-coenzyme A (CoA) in the mitochondrion. Metabolic labeling of the MPC mutants by isotopic glucose revealed impaired synthesis of acetyl-CoA, correlating with a global decrease in carbon flux through glycolysis and the tricarboxylic acid (TCA) cycle. Disruption of MPC proteins exerted only a modest effect on the parasite's virulence in mice, further highlighting its metabolic flexibility. In brief, our work reveals the modus operandi of pyruvate transport from the cytosol to the mitochondrion in the parasite, providing the missing link between glycolysis and the TCA cycle in T. gondii. IMPORTANCE T. gondii is a zoonotic parasite capable of infecting many warm-blooded organisms, including humans. Among others, a feature that allows it to parasitize multiple hosts is its exceptional metabolic plasticity. Although T. gondii can utilize different carbon sources, pyruvate homeostasis is critical for parasite growth. Pyruvate is produced primarily in the cytosol but metabolized in other organelles, such as the mitochondrion and apicoplast. The mechanism of import and physiological significance of pyruvate in these organelles remains unclear. Here, we identified the transporter of cytosol-derived pyruvate into the mitochondrion and studied its constituent subunits and their relevance. Our results show that cytosolic pyruvate is a major source of acetyl-CoA in the mitochondrion and that the mitochondrial pyruvate transporter is needed for optimal parasite growth. The mutants lacking the transporter are viable and virulent in a mouse model, underscoring the metabolic plasticity in the parasite's mitochondrion.
RESUMO
Toxoplasma gondii is a prevalent zoonotic pathogen infecting livestock as well as humans. The exceptional ability of this parasite to reproduce in several types of nucleated host cells necessitates a coordinated usage of endogenous and host-derived nutritional resources for membrane biogenesis. Phosphatidylethanolamine is the second most common glycerophospholipid in T. gondii, but how its requirement in the acutely-infectious fast-dividing tachyzoite stage is satisfied remains enigmatic. This work reveals that the parasite deploys de novo synthesis and salvage pathways to meet its demand for ester- and ether-linked PtdEtn. Auxin-mediated depletion of the phosphoethanolamine cytidylyltransferase (ECT) caused a lethal phenotype in tachyzoites due to impaired invasion and cell division, disclosing a vital role of the CDP-ethanolamine pathway during the lytic cycle. In accord, the inner membrane complex appeared disrupted concurrent with a decline in its length, parasite width and major phospholipids. Integrated lipidomics and isotope analyses of the TgECT mutant unveiled the endogenous synthesis of ester-PtdEtn, and salvage of ether-linked lipids from host cells. In brief, this study demonstrates how T. gondii operates various means to produce distinct forms of PtdEtn while featuring the therapeutic relevance of its de novo synthesis.
Assuntos
Toxoplasma , Humanos , Toxoplasma/genética , Toxoplasma/metabolismo , Fosfatidiletanolaminas/metabolismo , Éter/metabolismo , Glicerofosfolipídeos/metabolismo , Etil-Éteres/metabolismo , Éteres/metabolismoRESUMO
The ubiquitous pathogen Toxoplasma gondii has a complex lifestyle with different metabolic activities at different stages that are intimately linked to the parasitic environments. Here we identified the eukaryotic regulator of cellular homeostasis AMP-activated protein kinase (AMPK) in Toxoplasma and discovered its role in metabolic programming during parasite's lytic cycle. The catalytic subunit AMPKα is quickly phosphorylated after the release of intracellular parasites to extracellular environments, driving energy-producing catabolism to power parasite motility and invasion into host cells. Once inside host cells, AMPKα phosphorylation is reduced to basal level to promote a balance between energy production and biomass synthesis, allowing robust parasite replication. AMPKγ depletion abolishes AMPKα phosphorylation and suppresses parasite growth, which can be partially rescued by overexpressing wildtype AMPKα but not the phosphorylation mutants. Thus, through the cyclic reprogramming by AMPK, the parasites' metabolic needs at each stage are satisfied and the lytic cycle progresses robustly.
Assuntos
Parasitos , Toxoplasma , Animais , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Parasitos/metabolismo , Fosforilação , HomeostaseRESUMO
In the unprecedented single-cell sequencing and spatial multiomics era of biology, fluorescence in situ hybridization (FISH) technologies with higher sensitivity and robustness, especially for detecting short RNAs and other biomolecules, are greatly desired. Here, we develop the robust multiplex π-FISH rainbow method to detect diverse biomolecules (DNA, RNA, proteins, and neurotransmitters) individually or simultaneously with high efficiency. This versatile method is successfully applied to detect gene expression in different species, from microorganisms to plants and animals. Furthermore, we delineate the landscape of diverse neuron subclusters by decoding the spatial distribution of 21 marker genes via only two rounds of hybridization. Significantly, we combine π-FISH rainbow with hybridization chain reaction to develop π-FISH+ technology for short nucleic acid fragments, such as microRNA and prostate cancer anti-androgen therapy-resistant marker ARV7 splicing variant in circulating tumour cells from patients. Our study provides a robust biomolecule in situ detection technology for spatial multiomics investigation and clinical diagnosis.
Assuntos
MicroRNAs , Ácidos Nucleicos , Neoplasias da Próstata , Humanos , Masculino , Animais , Hibridização in Situ Fluorescente/métodos , MicroRNAs/genética , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/genéticaRESUMO
Toxoplasma gondii is an obligate intracellular zoonotic pathogen capable of infecting almost all cells of warm-blooded vertebrates. In intermediate hosts, this parasite reproduces asexually in two forms, the tachyzoite form during acute infection that proliferates rapidly and the bradyzoite form during chronic infection that grows slowly. Depending on the growth condition, the two forms can interconvert. The conversion of tachyzoites to bradyzoites is critical for T. gondii transmission, and the reactivation of persistent bradyzoites in intermediate hosts may lead to symptomatic toxoplasmosis. However, the mechanisms that control bradyzoite differentiation have not been well studied. Here, we review recent advances in the study of bradyzoite biology and stage conversion, aiming to highlight the determinants associated with bradyzoite development and provide insights to design better strategies for controlling toxoplasmosis.
RESUMO
Many apicomplexan parasites harbor a non-photosynthetic plastid called the apicoplast, which hosts important metabolic pathways like the methylerythritol 4-phosphate (MEP) pathway that synthesizes isoprenoid precursors. Yet many details in apicoplast metabolism are not well understood. In this study, we examined the physiological roles of four glycolytic enzymes in the apicoplast of Toxoplasma gondii. Many glycolytic enzymes in T. gondii have two or more isoforms. Endogenous tagging each of these enzymes found that four of them were localized to the apicoplast, including pyruvate kinase2 (PYK2), phosphoglycerate kinase 2 (PGK2), triosephosphate isomerase 2 (TPI2) and phosphoglyceraldehyde dehydrogenase 2 (GAPDH2). The ATP generating enzymes PYK2 and PGK2 were thought to be the main energy source of the apicoplast. Surprisingly, deleting PYK2 and PGK2 individually or simultaneously did not cause major defects on parasite growth or virulence. In contrast, TPI2 and GAPDH2 are critical for tachyzoite proliferation. Conditional depletion of TPI2 caused significant reduction in the levels of MEP pathway intermediates and led to parasite growth arrest. Reconstitution of another isoprenoid precursor synthesis pathway called the mevalonate pathway in the TPI2 depletion mutant partially rescued its growth defects. Similarly, knocking down the GAPDH2 enzyme that produces NADPH also reduced isoprenoid precursor synthesis through the MEP pathway and inhibited parasite proliferation. In addition, it reduced de novo fatty acid synthesis in the apicoplast. Together, these data suggest a model that the apicoplast dwelling TPI2 provides carbon source for the synthesis of isoprenoid precursor, whereas GAPDH2 supplies reducing power for pathways like MEP, fatty acid synthesis and ferredoxin redox system in T. gondii. As such, both enzymes are critical for parasite growth and serve as potential targets for anti-toxoplasmic intervention designs. On the other hand, the dispensability of PYK2 and PGK2 suggest additional sources for energy in the apicoplast, which deserves further investigation.
Assuntos
Apicoplastos , Parasitos , Toxoplasma , Animais , Toxoplasma/metabolismo , Redes e Vias Metabólicas , Parasitos/metabolismo , Ácido Pirúvico/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
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.
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Parasitos , Toxoplasma , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Ciclo Celular , Divisão Celular , Humanos , Parasitos/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
Toxoplasma gondii (T. gondii) is a pathogen belonging to the apicomplexan phylum, and it threatens human and animal health. Calcium ions, a critical second messenger in cells, can regulate important biological processes, including parasite invasion and egress. Calmodulin (CaM) is a small, highly conserved, Ca2+-binding protein found in all eukaryotic cells. After binding to Ca2+, CaM can be activated to interact with various proteins. However, little is known about CaM's function and its interacting proteins in T. gondii. In this study, we successfully knocked down CaM in the T. gondii parent strain TATI using a tetracycline-off system with the Toxoplasma CaM promoter. The results indicated that CaM was required for tachyzoite proliferation, invasion, and egress and that CaM depletion resulted in apicoplast loss, thus threatening parasite survival in the next lytic cycle. In the tachyzoite stage, CaM loss caused significant anomalies in the parasite's basal constriction, motility, and parasite rosette-like arrangement in the parasitophorous vacuole (PV). These phenotypic defects caused by CaM depletion indicate the importance of CaM in T. gondii. Therefore, it is important to identify the CaM-interacting proteins in T. gondii. Applying BioID technology, more than 300 CaM's proximal interacting proteins were identified from T. gondii. These CaM partners were broadly distributed throughout the parasite. Furthermore, the protein interactome and transcriptome analyses indicated the potential role of CaM in ion binding, cation binding, metal ion binding, calcium ion binding, and oxidation-reduction. Our findings shed light on the CaM function and CaM-interactome in T. gondii and other eukaryotes. IMPORTANCE Toxoplasma gondii is an intracellular pathogen that threatens human and animal health. This unicellular parasite is active in many biological processes, such as egress and invasion. The implementation efficiency of T. gondii biological processes is dependent on signal transmission. Ca2+, as a second messenger, is essential for the parasite's life cycle. Calmodulin, a ubiquitous Ca2+ receptor protein, is highly conserved and mediates numerous Ca2+-dependent events in eukaryotes. Few CaM functions or regulated partners have been characterized in T. gondii tachyzoites. Here, we reported the essential functions of calmodulin in T. gondii tachyzoite and the identification of its interacting partners using BioID technology, shedding light on the CaM function and CaM-interactome in Toxoplasma gondii and other eukaryotes.
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Parasitos , Toxoplasma , Animais , Humanos , Toxoplasma/genética , Calmodulina/genética , Calmodulina/metabolismo , Cálcio/metabolismo , Tecnologia , Tetraciclinas/metabolismo , Cátions/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
BACKGROUND: It has been reported that the NF-κB pathway, an important component of host defense system against pathogens infections, can be differentially modulated by different Toxoplasma gondii strains, depending on the polymorphism of the GRA15 protein. The recently isolated Toxoplasma strain T.gHB1 is a type 1 (ToxoDB#10) strain but shows different virulence determination mechanisms compared to the classic type 1 strains like RH (ToxoDB#10). Therefore, it is worth investigating whether the T.gHB1 strain (ToxoDB#10) affects the host NF-κB signaling pathway. METHODS: The effects of T.gHB1 (ToxoDB#10) on host NF-κB pathway were investigated in HEK293T cells. The GRA15 gene product was analyzed by bioinformatics, and its effect on NF-κB activation was examined by Western blotting and nuclear translocation of p65. Different truncations of T.gHB1 GRA15 were constructed to map the critical domains for NF-κB activation. RESULTS: We demonstrated that the NF-κB pathway signaling pathway could be activated by the newly identified type 1 T.gHB1 strain (ToxoDB#10) of Toxoplasma, while the classic type 1 strain RH (ToxoDB#10) did not. T.gHB1 GRA15 possesses only one transmembrane region with an extended C terminal region, which is distinct from that of classic type 1 (ToxoDB#10) and type 2 (ToxoDB#1) strains. T.gHB1 GRA15 could clearly induce IκBα phosphorylation and p65 nuclear translocation. Dual luciferase assays in HEK293T cells revealed a requirement for 194-518 aa of T.gHB1 GRA15 to effectively activate NF-κB. CONCLUSIONS: The overall results indicated that the newly isolated type 1 isolate T.gHB1 (ToxoDB#10) had a unique GRA15, which could activate the host NF-κB signaling through inducing IκBα phosphorylation and p65 nuclear translocation. These results provide new insights for our understanding of the interaction between Toxoplasma parasites and its hosts.
Assuntos
NF-kappa B , Proteínas de Protozoários , Toxoplasma , Células HEK293 , Humanos , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Transdução de SinaisRESUMO
Metabolic pathways underpin the growth and virulence of intracellular parasites and are therefore promising antiparasitic targets. The pentose phosphate pathway (PPP) is vital in most organisms, providing a reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) and ribose sugar for nucleotide synthesis; however, it has not yet been studied in Toxoplasma gondii, a widespread intracellular pathogen and a model protozoan organism. Herein, we show that T. gondii has a functional PPP distributed in the cytoplasm and nucleus of its acutely-infectious tachyzoite stage. We produced eight parasite mutants disrupting seven enzymes of the PPP in T. gondii. Our data show that of the seven PPP proteins, the two glucose-6-phosphate dehydrogenases (TgG6PDH1, TgG6PDH2), one of the two 6-phosphogluconate dehydrogenases (Tg6PGDH1), ribulose-5-phosphate epimerase (TgRuPE) and transaldolase (TgTAL) are dispensable in vitro as well as in vivo, disclosing substantial metabolic plasticity in T. gondii. Among these, TgG6PDH2 plays a vital role in defense against oxidative stress by the pathogen. Further, we show that Tg6PGDH2 and ribulose-5-phosphate isomerase (TgRPI) are critical for tachyzoite growth. The depletion of TgRPI impairs the flux of glucose in central carbon pathways, and causes decreased expression of ribosomal, microneme and rhoptry proteins. In summary, our results demonstrate the physiological need of the PPP in T. gondii while unraveling metabolic flexibility and antiparasitic targets.
Assuntos
Via de Pentose Fosfato , Toxoplasma , Antiparasitários , Carbono/metabolismo , Glucose/metabolismo , Glucose-6-Fosfato/metabolismo , Isomerases/metabolismo , NADP/metabolismo , Via de Pentose Fosfato/fisiologia , Fosfatos/metabolismo , Racemases e Epimerases/metabolismo , Ribose , Toxoplasma/metabolismo , Transaldolase/metabolismoRESUMO
Toxoplasma gondii is an obligate intracellular parasite that acquires all necessary nutrients from the hosts, but the exact nutrient acquisition mechanisms are poorly understood. Here, we identified three putative phosphate transporters in T. gondii. TgPiT and TgPT2 are mainly on the plasma membrane, whereas TgmPT is localized to the mitochondrion. TgPiT and TgmPT are widely present and conserved in apicomplexan parasites that include Plasmodium and Eimeria species. Nonetheless, they are dispensable for the growth and virulence of Toxoplasma. TgPT2, on the other hand, is restricted to coccidia parasites and is essential for Toxoplasma survival. TgPT2 depletion led to reduced motility and invasion, as well as growth arrest of the parasites both in vitro and in vivo. Both TgPiT and TgPT2 have phosphate transport activities and contribute to parasites' inorganic phosphate (Pi) absorption. Interestingly, the Pi importing activity of Toxoplasma parasites could be competitively inhibited by ATP and AMP. Furthermore, direct uptake of 32P-ATP was also observed, indicating the parasites' ability to scavenge host ATP. Nonetheless, ATP/AMP import is not mediated by TgPiT or TgPT2, suggesting additional mechanisms. Together, these results show the complex pathways of phosphate transport in Toxoplasma, and TgPT2 is a potential target for antitoxoplasmic intervention design due to its essential role in parasite growth. IMPORTANCE To grow and survive within host cells, Toxoplasma must scavenge necessary nutrients from hosts to support its parasitism. Transporters located in the plasma membrane of the parasites play critical roles in nutrient acquisition. Toxoplasma encodes a large number of transporters, but so far, only a few have been characterized. In this study, we identified two phosphate transporters, TgPiT and TgPT2, to localize to the plasma membrane of Toxoplasma. Although both TgPiT and TgPT2 possess phosphate transport activities, only the novel transporter TgPT2 was essential for parasite growth, both in vitro and in vivo. In addition, TgPT2 and its orthologs are only present in coccidia parasites. As such, TgPT2 represents a potential target for drug design against toxoplasmosis. In addition, our data indicated that Toxoplasma can take up ATP and AMP from the environment, providing new insights into the energy metabolism of Toxoplasma.