RESUMO
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
Glycerophospholipids have emerged as a significant contributor to the intracellular growth of pathogenic protist Toxoplasma gondii. Phosphatidylserine (PtdSer) is one such lipid, attributed to the locomotion and motility-dependent invasion and egress events in its acutely infectious tachyzoite stage. However, the de novo synthesis of PtdSer and the importance of the pathway in tachyzoites remain poorly understood. We show that a base-exchange-type PtdSer synthase (PSS) located in the parasite's endoplasmic reticulum produces PtdSer, which is rapidly converted to phosphatidylethanolamine (PtdEtn) by PtdSer decarboxylase (PSD) activity. The PSS-PSD pathway enables the synthesis of several lipid species, including PtdSer (16:0/18:1) and PtdEtn (18:2/20:4, 18:1/18:2 and 18:2/22:5). The PSS-depleted strain exhibited a lower abundance of the major ester-linked PtdEtn species and concurrent accrual of host-derived ether-PtdEtn species. Most phosphatidylthreonine (PtdThr) species-an exclusive natural analog of PtdSer, also made in the endoplasmic reticulum-were repressed. PtdSer species, however, remained largely unaltered, likely due to the serine-exchange reaction of PtdThr synthase in favor of PtdSer upon PSS depletion. Not least, the loss of PSS abrogated the lytic cycle of tachyzoites, impairing the cell division, motility, and egress. In a nutshell, our data demonstrate a critical role of PSS in the biogenesis of PtdSer and PtdEtn species and its physiologically essential repurposing for the asexual reproduction of a clinically relevant intracellular pathogen.
Assuntos
Retículo Endoplasmático , Toxoplasma , Toxoplasma/enzimologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/enzimologia , Humanos , Fosfatidilserinas/metabolismo , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/metabolismo , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , CarboxiliasesRESUMO
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
Lipid flipping in the membrane bilayers is a widespread eukaryotic phenomenon that is catalyzed by assorted P4-ATPases. Its occurrence, mechanism, and importance in apicomplexan parasites have remained elusive, however. Here we show that Toxoplasma gondii, an obligate intracellular parasite with high clinical relevance, can salvage phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) but not phosphatidylcholine (PtdCho) probes from its milieu. Consistently, the drug analogs of PtdCho are broadly ineffective in the parasite culture. NBD-PtdSer imported to the parasite interior is decarboxylated to NBD-PtdEtn, while the latter is not methylated to yield PtdCho, which confirms the expression of PtdSer decarboxylase but a lack of PtdEtn methyltransferase activity and suggests a role of exogenous lipids in membrane biogenesis of T. gondii. Flow cytometric quantitation of NBD-probes endorsed the selectivity of phospholipid transport and revealed a dependence of the process on energy and protein. Accordingly, our further work identified five P4-ATPases (TgP4-ATPase1-5), all of which harbor the signature residues and motifs required for phospholipid flipping. Of the four proteins expressed during the lytic cycle, TgP4-ATPase1 is present in the apical plasmalemma; TgP4-ATPase3 resides in the Golgi network along with its noncatalytic partner Ligand Effector Module 3 (TgLem3), whereas TgP4-ATPase2 and TgP4-ATPase5 localize in the plasmalemma as well as endo/cytomembranes. Last but not least, auxin-induced degradation of TgP4-ATPase1-3 impaired the parasite growth in human host cells, disclosing their crucial roles during acute infection. In conclusion, we show selective translocation of PtdEtn and PtdSer at the parasite surface and provide the underlying mechanistic and physiological insights in a model eukaryotic pathogen.
Assuntos
Adenosina Trifosfatases/genética , Bicamadas Lipídicas/metabolismo , Toxoplasma/genética , Toxoplasmose/genética , Adenosina Trifosfatases/química , Membrana Celular/genética , Membrana Celular/metabolismo , Citometria de Fluxo , Glicerofosfolipídeos/metabolismo , Complexo de Golgi/química , Complexo de Golgi/enzimologia , Humanos , Bicamadas Lipídicas/química , Lipídeos/química , Lipídeos/genética , Fosfatidilcolinas/genética , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/genética , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Toxoplasma/enzimologia , Toxoplasma/patogenicidade , Toxoplasmose/parasitologiaRESUMO
Toxoplasma gondii is a common protozoan parasite that infects a wide range of hosts, including livestock and humans. Previous studies have suggested that the type 2 fatty acid synthesis (FAS2) pathway, located in the apicoplast (a nonphotosynthetic plastid relict), is crucial for the parasite's survival. Here we examined the physiological relevance of fatty acid synthesis in T. gondii by focusing on the pyruvate dehydrogenase complex and malonyl-CoA-[acyl carrier protein] transacylase (FabD), which are located in the apicoplast to drive de novo fatty acid biosynthesis. Our results disclosed unexpected metabolic resilience of T. gondii tachyzoites, revealing that they can tolerate CRISPR/Cas9-assisted genetic deletions of three pyruvate dehydrogenase subunits or FabD. All mutants were fully viable in prolonged cultures, albeit with impaired growth and concurrent loss of the apicoplast. Even more surprisingly, these mutants displayed normal virulence in mice, suggesting an expendable role of the FAS2 pathway in vivo Metabolic labeling of the Δpdh-e1α mutant showed reduced incorporation of glucose-derived carbon into fatty acids with medium chain lengths (C14:0 and C16:0), revealing that FAS2 activity was indeed compromised. Moreover, supplementation of exogenous C14:0 or C16:0 significantly reversed the growth defect in the Δpdh-e1α mutant, indicating salvage of these fatty acids. Together, these results demonstrate that the FAS2 pathway is dispensable during the lytic cycle of Toxoplasma because of its remarkable flexibility in acquiring fatty acids. Our findings question the long-held assumption that targeting this pathway has significant therapeutic potential for managing Toxoplasma infections.
Assuntos
Apicoplastos/metabolismo , Ácidos Graxos/metabolismo , Ácidos Graxos/farmacologia , Toxoplasma/metabolismo , Proteína de Transporte de Acila S-Maloniltransferase/genética , Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Apicoplastos/genética , Ácidos Graxos/genética , Deleção de Genes , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/genéticaRESUMO
The birth and subsequent evolution of optogenetics has resulted in an unprecedented advancement in our understanding of the brain. Its outstanding success does usher wider applications; however, the tool remains still largely relegated to neuroscience. Here, we introduce selected aspects of optogenetics with potential applications in infection biology that will not only answer long-standing questions about intracellular pathogens (parasites, bacteria, viruses) but also broaden the dimension of current research in entwined models. In this essay, we illustrate how a judicious integration of optogenetics with routine methods can illuminate the host-pathogen interactions in a way that has not been feasible otherwise.
Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Optogenética/métodos , Animais , HumanosRESUMO
Toxoplasma gondii is a globally prevalent parasitic protist. It is well-known for its ability to infect almost all nucleated vertebrate cells, which is reflected by its unique metabolic architecture. Its fast-growing tachyzoite stage catabolizes glucose via glycolysis to yield l-lactate as a major by-product that must be exported from the cell to prevent toxicity; the underlying mechanism remains to be elucidated, however. Herein, we report three formate-nitrite transporter (FNT)-type monocarboxylate/proton symporters located in the plasma membrane of the T. gondii tachyzoite stage. We observed that all three proteins transport both l-lactate and formate in a pH-dependent manner and are inhibited by 2-hydroxy-chromanones (a class of small synthetic molecules). We also show that these compounds pharmacologically inhibit T. gondii growth. Using a chemical biology approach, we identified the critical residues in the substrate-selectivity region of the parasite transporters that determine differential specificity and sensitivity toward both substrates and inhibitors. Our findings further indicate that substrate specificity in FNT family proteins from T. gondii has evolved such that a functional repurposing of prokaryotic-type transporters helps fulfill a critical metabolic role in a clinically important parasitic protist. In summary, we have identified and characterized the lactate transporters of T. gondii and have shown that compounds blocking the FNTs in this parasite can inhibit its growth, suggesting that these transporters could have utility as potential drug targets.
Assuntos
Antiprotozoários/química , Membrana Celular , Transportadores de Ácidos Monocarboxílicos , Proteínas de Protozoários , Toxoplasma , Sítios de Ligação , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/química , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Especificidade por Substrato , Toxoplasma/genética , Toxoplasma/metabolismoRESUMO
Toxoplasma gondii is considered to be one of the most successful intracellular pathogens, because it can reproduce in varied nutritional milieus, encountered in diverse host cell types of essentially any warm-blooded organism. Our earlier work demonstrated that the acute (tachyzoite) stage of T. gondii depends on cooperativity of glucose and glutamine catabolism to meet biosynthetic demands. Either of these two nutrients can sustain the parasite survival; however, what determines the metabolic plasticity has not yet been resolved. Here, we reveal two discrete phosphoenolpyruvate carboxykinase (PEPCK) enzymes in the parasite, one of which resides in the mitochondrion (TgPEPCKmt), whereas the other protein is not expressed in tachyzoites (TgPEPCKnet). Parasites with an intact glycolysis can tolerate genetic deletions of TgPEPCKmt as well as of TgPEPCKnet, indicating their nonessential roles for tachyzoite survival. TgPEPCKnet can also be ablated in a glycolysis-deficient mutant, while TgPEPCKmt is refractory to deletion. Consistent with this, the lytic cycle of a conditional mutant of TgPEPCKmt in the glycolysis-impaired strain was aborted upon induced repression of the mitochondrial isoform, demonstrating its essential role for the glucose-independent survival of parasites. Isotope-resolved metabolomics of the conditional mutant revealed defective flux of glutamine-derived carbon into RNA-bound ribose sugar as well as metabolites associated with gluconeogenesis, entailing a critical nodal role of PEPCKmt in linking catabolism of glucose and glutamine with anabolic pathways. Our data also suggest a homeostatic function ofTgPEPCKmt in cohesive operation of glycolysis and the tricarboxylic acid cycle in a normal glucose-replete milieu. Conversely, we found that the otherwise integrative enzyme pyruvate carboxylase (TgPyC) is dispensable not only in glycolysis-competent but also in glycolysis-deficient tachyzoites despite a mitochondrial localization. Last but not least, the observed physiology of T. gondii tachyzoites appears to phenocopy cancer cells, which holds promise for developing common therapeutics against both threats.
Assuntos
Mitocôndrias/enzimologia , Modelos Biológicos , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Ciclo do Ácido Cítrico , Deleção de Genes , Gluconeogênese , Glucose/metabolismo , Glutamina/metabolismo , Glicólise , Homeostase , Isoenzimas/genética , Isoenzimas/metabolismo , Metabolômica/métodos , Viabilidade Microbiana , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Mutação , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Piruvato Carboxilase/genética , Piruvato Carboxilase/metabolismo , Proteínas Recombinantes de Fusão , Ribose/biossíntese , Toxoplasma/citologia , Toxoplasma/crescimento & desenvolvimentoRESUMO
Toxoplasma gondii is among the most prevalent protozoan parasites, which infects a wide range of organisms, including one-third of the human population. Its rapid intracellular replication within a vacuole requires efficient synthesis of glycerophospholipids. Cytidine diphosphate-diacylglycerol (CDP-DAG) serves as a major precursor for phospholipid synthesis. Given the peculiarities of lipid biogenesis, understanding the mechanism and physiological importance of CDP-DAG synthesis is particularly relevant in T. gondii Here, we report the occurrence of two phylogenetically divergent CDP-DAG synthase (CDS) enzymes in the parasite. The eukaryotic-type TgCDS1 and the prokaryotic-type TgCDS2 reside in the endoplasmic reticulum and apicoplast, respectively. Conditional knockdown of TgCDS1 severely attenuated the parasite growth and resulted in a nearly complete loss of virulence in a mouse model. Moreover, mice infected with the TgCDS1 mutant became fully resistant to challenge infection with a hyper-virulent strain of T. gondii The residual growth of the TgCDS1 mutant was abolished by consecutive deletion of TgCDS2. Lipidomic analyses of the two mutants revealed significant and specific declines in phosphatidylinositol and phosphatidylglycerol levels upon repression of TgCDS1 and after deletion of TgCDS2, respectively. Our data suggest a "division of labor" model of lipid biogenesis in T. gondii in which two discrete CDP-DAG pools produced in the endoplasmic reticulum and apicoplast are subsequently used for the synthesis of phosphatidylinositol in the Golgi bodies and phosphatidylglycerol in the mitochondria. The essential and divergent nature of CDP-DAG synthesis in the parasite apicoplast offers a potential drug target to inhibit the asexual reproduction of T. gondii.
Assuntos
Diacilglicerol Colinofosfotransferase/genética , Glicerofosfolipídeos/biossíntese , Proteínas de Protozoários/genética , Toxoplasma/enzimologia , Animais , Animais Geneticamente Modificados , Apicoplastos/enzimologia , Diacilglicerol Colinofosfotransferase/metabolismo , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Deleção de Genes , Complexo de Golgi/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Mutação , Fosfatidilgliceróis/química , Fosfatidilinositóis/química , Filogenia , Domínios Proteicos , Proteínas de Protozoários/metabolismo , Toxoplasma/genética , VirulênciaRESUMO
Coccidiosis, caused by different species of Eimeria parasites, is an economically important disease of poultry and livestock worldwide. Here we report previously unknown alterations in the gut microbes and metabolism of BALB/c mice infected with Eimeria falciformis Specifically, we observed a significant shift in the abundance of cecal bacteria and disrupted metabolism in parasitized animals. The relative abundances of Lachnospiraceae bacterium NK4A136, Ruminiclostridium, Alistipes, and Lactobacillus declined in response to E. falciformis infection, whereas Escherichia, Shigella, Helicobacter, Klebsiella, and Bacteroides were increased. Carbohydrate and amino acid metabolites in the serum samples of infected mice were significantly altered compared to naïve controls. Levels of amino acids, including asparagine, histidine, l-cysteine, tryptophan, lysine, glycine, serine, alanine, proline, ornithine, methionine, and valine, decreased on day 7 postinfection before returning to baseline on day 14. In addition, increased levels of indolelactate and mannitol and a reduced amount of oxalic acid indicated impaired carbon metabolism upon parasitic infection. These data demonstrate that intestinal coccidial infection perturbs the microbiota and disrupts carbon and nitrogen metabolism.
Assuntos
Coccidiose/fisiopatologia , Eimeria/patogenicidade , Microbioma Gastrointestinal/fisiologia , Interações Hospedeiro-Parasita/fisiologia , Redes e Vias Metabólicas/fisiologia , Animais , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The major membrane phospholipid classes, described thus far, include phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), and phosphatidylinositol (PtdIns). Here, we demonstrate the natural occurrence and genetic origin of an exclusive and rather abundant lipid, phosphatidylthreonine (PtdThr), in a common eukaryotic model parasite, Toxoplasma gondii. The parasite expresses a novel enzyme PtdThr synthase (TgPTS) to produce this lipid in its endoplasmic reticulum. Genetic disruption of TgPTS abrogates de novo synthesis of PtdThr and impairs the lytic cycle and virulence of T. gondii. The observed phenotype is caused by a reduced gliding motility, which blights the parasite egress and ensuing host cell invasion. Notably, the PTS mutant can prevent acute as well as yet-incurable chronic toxoplasmosis in a mouse model, which endorses its potential clinical utility as a metabolically attenuated vaccine. Together, the work also illustrates the functional speciation of two evolutionarily related membrane phospholipids, i.e., PtdThr and PtdSer.
Assuntos
Retículo Endoplasmático/enzimologia , Glicerofosfolipídeos/metabolismo , Proteínas de Protozoários/metabolismo , Treonina/análogos & derivados , Toxoplasma/fisiologia , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Animais , Encéfalo/imunologia , Encéfalo/parasitologia , Encéfalo/patologia , Células Cultivadas , Retículo Endoplasmático/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Organismos Geneticamente Modificados/imunologia , Organismos Geneticamente Modificados/metabolismo , Encistamento de Parasitas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Vacinas Protozoárias/uso terapêutico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Pele/citologia , Pele/imunologia , Pele/metabolismo , Pele/parasitologia , Treonina/metabolismo , Toxoplasma/genética , Toxoplasma/imunologia , Toxoplasma/patogenicidade , Toxoplasmose/imunologia , Toxoplasmose/parasitologia , Toxoplasmose/patologia , Toxoplasmose/prevenção & controle , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Vacinas Atenuadas/uso terapêutico , VirulênciaRESUMO
Toxoplasma gondii is a widespread protozoan parasite infecting nearly all warm-blooded organisms. Asexual reproduction of the parasite within its host cells is achieved by consecutive lytic cycles, which necessitates biogenesis of significant energy and biomass. Here we show that glucose and glutamine are the two major physiologically important nutrients used for the synthesis of macromolecules (ATP, nucleic acid, proteins, and lipids) in T. gondii, and either of them is sufficient to ensure the parasite survival. The parasite can counteract genetic ablation of its glucose transporter by increasing the flux of glutamine-derived carbon through the tricarboxylic acid cycle and by concurrently activating gluconeogenesis, which guarantee a continued biogenesis of ATP and biomass for host-cell invasion and parasite replication, respectively. In accord, a pharmacological inhibition of glutaminolysis or oxidative phosphorylation arrests the lytic cycle of the glycolysis-deficient mutant, which is primarily a consequence of impaired invasion due to depletion of ATP. Unexpectedly, however, intracellular parasites continue to proliferate, albeit slower, notwithstanding a simultaneous deprivation of glucose and glutamine. A growth defect in the glycolysis-impaired mutant is caused by a compromised synthesis of lipids, which cannot be counterbalanced by glutamine but can be restored by acetate. Consistently, supplementation of parasite cultures with exogenous acetate can amend the lytic cycle of the glucose transport mutant. Such plasticity in the parasite's carbon flux enables a growth-and-survival trade-off in assorted nutrient milieus, which may underlie the promiscuous survival of T. gondii tachyzoites in diverse host cells. Our results also indicate a convergence of parasite metabolism with cancer cells.
Assuntos
Glucose/metabolismo , Glutamina/metabolismo , Estágios do Ciclo de Vida , Parasitos/crescimento & desenvolvimento , Parasitos/metabolismo , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/metabolismo , Acetatos/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Biomassa , Metabolismo dos Carboidratos/efeitos dos fármacos , Carbono/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Metabolismo Energético/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/parasitologia , Glicólise/efeitos dos fármacos , Humanos , Espaço Intracelular/parasitologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Lipídeos/química , Masculino , Modelos Biológicos , Mutação/genética , Fosforilação Oxidativa/efeitos dos fármacos , Parasitos/efeitos dos fármacos , Fenótipo , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Toxoplasma/efeitos dos fármacos , Toxoplasmose/metabolismo , Toxoplasmose/parasitologiaRESUMO
Toxoplasma gondii is a highly prevalent obligate intracellular parasite of the phylum Apicomplexa, which also includes other parasites of clinical and/or veterinary importance, such as Plasmodium, Cryptosporidium, and Eimeria. Acute infection by Toxoplasma is hallmarked by rapid proliferation in its host cells and requires a significant synthesis of parasite membranes. Phosphatidylethanolamine (PtdEtn) is the second major phospholipid class in T. gondii. Here, we reveal that PtdEtn is produced in the parasite mitochondrion and parasitophorous vacuole by decarboxylation of phosphatidylserine (PtdSer) and in the endoplasmic reticulum by fusion of CDP-ethanolamine and diacylglycerol. PtdEtn in the mitochondrion is synthesized by a phosphatidylserine decarboxylase (TgPSD1mt) of the type I class. TgPSD1mt harbors a targeting peptide at its N terminus that is required for the mitochondrial localization but not for the catalytic activity. Ablation of TgPSD1mt expression caused up to 45% growth impairment in the parasite mutant. The PtdEtn content of the mutant was unaffected, however, suggesting the presence of compensatory mechanisms. Indeed, metabolic labeling revealed an increased usage of ethanolamine for PtdEtn synthesis by the mutant. Likewise, depletion of nutrients exacerbated the growth defect (â¼56%), which was partially restored by ethanolamine. Besides, the survival and residual growth of the TgPSD1mt mutant in the nutrient-depleted medium also indicated additional routes of PtdEtn biogenesis, such as acquisition of host-derived lipid. Collectively, the work demonstrates a metabolic cooperativity between the parasite organelles, which ensures a sustained lipid synthesis, survival and growth of T. gondii in varying nutritional milieus.
Assuntos
Carboxiliases/metabolismo , Mitocôndrias/metabolismo , Fosfatidiletanolaminas/biossíntese , Proteínas de Protozoários/metabolismo , Toxoplasma/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Carboxiliases/classificação , Carboxiliases/genética , Sobrevivência Celular , Cistina Difosfato/análogos & derivados , Cistina Difosfato/metabolismo , Diglicerídeos/metabolismo , Etanolaminas/metabolismo , Dados de Sequência Molecular , Filogenia , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasma/metabolismoRESUMO
BACKGROUND: cAMP research in intracellular parasites remains underappreciated, and it requires a specific method for cyclic nucleotide regulation. RESULTS: Optogenetic induction of cAMP in T. gondii affects host-cell invasion, stage-specific expression, and parasite differentiation. The underlying method allows a versatile control of parasite cAMP. CONCLUSIONS: Optogenetic parasite strains offer valuable tools for dissecting cAMP-mediated processes. SIGNIFICANCE: The method is applicable to other gene-tractable intertwined systems. Successful infection and transmission of the obligate intracellular parasite Toxoplasma gondii depends on its ability to switch between fast-replicating tachyzoite (acute) and quiescent bradyzoite (chronic) stages. Induction of cAMP in the parasitized host cells has been proposed to influence parasite differentiation. It is not known whether the parasite or host cAMP is required to drive this phenomenon. Other putative roles of cAMP for the parasite biology also remain to be identified. Unequivocal research on cAMP-mediated signaling in such intertwined systems also requires a method for an efficient and spatial control of the cAMP pool in the pathogen or in the enclosing host cell. We have resolved these critical concerns by expressing a photoactivated adenylate cyclase that allows light-sensitive control of the parasite or host-cell cAMP. Using this method, we reveal multiple roles of the parasite-derived cAMP in host-cell invasion, stage-specific expression, and asexual differentiation. An optogenetic method provides many desired advantages such as: (i) rapid, transient, and efficient cAMP induction in extracellular/intracellular and acute/chronic stages; (ii) circumvention of the difficulties often faced in cultures, i.e. poor diffusion, premature degradation, steady activation, and/or pleiotropic effects of cAMP agonists and antagonists; (iii) genetically encoded enzyme expression, thus inheritable to the cell progeny; and (iv) conditional and spatiotemporal control of cAMP levels. Importantly, a successful optogenetic application in Toxoplasma also illustrates its wider utility to study cAMP-mediated signaling in other genetically amenable two-organism systems such as in symbiotic and pathogen-host models.
Assuntos
Adenilil Ciclases/biossíntese , AMP Cíclico/fisiologia , Toxoplasma/metabolismo , Adenilil Ciclases/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/parasitologia , Regulação Enzimológica da Expressão Gênica , Interações Hospedeiro-Parasita , Humanos , Optogenética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Sistemas do Segundo Mensageiro , Toxoplasma/genética , Toxoplasma/fisiologiaRESUMO
Dynein light chain 8 (DLC8) is a ubiquitous eukaryotic protein regulating diverse cellular functions. We show that the obligate intracellular parasite Toxoplasma gondii harbors 4 DLC8 proteins (TgDLC8a-d), of which only TgDLC8a clusters in the mainstream LC8 class. TgDLC8b-d proteins form a divergent and alveolate-specific clade. TgDLC8b-d proteins are largely cytosolic, whereas TgDLC8a resides in the conoid at the apical end of T. gondii. The apical location of TgDLC8a is also not shared by its nearly identical Eimeria (EtDLC8a), Plasmodium (PfDLC8), or human (HsDLC8) orthologs. Notwithstanding an exclusive conoid targeting, TgDLC8a exhibits a classical LC8 structure. It forms a homodimer by swapping of the ß strands that interact with the antiparallel ß' strands of the opposing monomers. The TgDLC8a dimer contains two identical binding grooves and appears to be adapted for multitarget recognition. By contrast, the previously reported PfDLC8 homodimer is shaped by binding of the ß strand with the parallel ß' strand and lacks such a distinct binding interface. Our comparisons suggest an unexpected structural and functional divergence of the two otherwise conserved proteins from apicomplexan parasites. Finally, we demonstrate that a phosphomimetic S88E mutation renders the TgDLC8a-S88E mutant monomeric and cytosolic in T. gondii, and its overexpression inhibits the parasite growth in human fibroblasts.
Assuntos
Dineínas/metabolismo , Multimerização Proteica , Proteínas de Protozoários/metabolismo , Toxoplasma/enzimologia , Toxoplasma/crescimento & desenvolvimento , Sequência de Aminoácidos , Substituição de Aminoácidos , Células Cultivadas , Dineínas/genética , Fibroblastos/parasitologia , Fibroblastos/patologia , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas de Protozoários/genética , Toxoplasma/genéticaRESUMO
Ribose-5-phosphate (R5P) is a precursor for nucleic acid biogenesis; however, the importance and homeostasis of R5P in the intracellular parasite Toxoplasma gondii remain enigmatic. Here, we show that the cytoplasmic sedoheptulose-1,7-bisphosphatase (SBPase) is dispensable. Still, its co-deletion with transaldolase (TAL) impairs the double mutant's growth and increases 13C-glucose-derived flux into pentose sugars via the transketolase (TKT) enzyme. Deletion of the latter protein affects the parasite's fitness but is not lethal and is correlated with an increased carbon flux via the oxidative pentose phosphate pathway. Further, loss of TKT leads to a decline in 13C incorporation into glycolysis and the TCA cycle, resulting in a decrease in ATP levels and the inability of phosphoribosyl-pyrophosphate synthetase (PRPS) to convert R5P into 5'-phosphoribosyl-pyrophosphate and thereby contribute to the production of AMP and IMP. Likewise, PRPS is essential for the lytic cycle. Not least, we show that RuPE-mediated metabolic compensation is imperative for the survival of the ΔsbpaseΔtal strain. In conclusion, we demonstrate that multiple routes can flexibly supply R5P to enable parasite growth and identify catalysis by TKT and PRPS as critical enzymatic steps. Our work provides novel biological and therapeutic insights into the network design principles of intracellular parasitism in a clinically-relevant pathogen.
Assuntos
Toxoplasma , Toxoplasma/metabolismo , Difosfatos/metabolismo , Ribosemonofosfatos/metabolismo , Glicólise , Via de Pentose FosfatoRESUMO
Anticoccidial vaccines comprising living oocysts of Eimeria tenella, Eimeria necatrix, Eimeria maxima, and Eimeria acervulina are used to control coccidiosis. This study explored the potential of IL-1ß to act as a molecular adjuvant for enhancing the immunogenicity of Eimeria necatrix and mucosal immunity. We engineered E. necatrix to express a functional chIL-1ß (EnIL-1ß) and immunized chickens with oocysts of the wild type (EnWT) and tranegenic (EnIL-1ß) strains, respectively. The chickens were then challenged with EnWT oocysts to examine the immunogenicity-enhancing potential of chIL-1ß. As expected, the oocyst output of EnIL-1ß-immunized chickens was significantly reduced compared to those immunized using EnWT. No difference in body weight gain and lesion scores of EnIL-1ß and EnWT groups was observed. The parasite load in the small intestine and caeca showed that the invasion and replication of EnIL-1ß was not affected. However, the markers of immunogenicity and mucosal barrier, Claudin-1 and avian ß-defensin-1, were elevated in EnIL-1ß-infected chickens. Ectopic expression of chIL-1ß in E. necatrix thus appears to improve its immunogenicity and mucosal immunity, without increasing pathogenicity. Our findings support chIL-1ß as a candidate for development of effective live-oocyst-based anticoccidial vaccines.
Assuntos
Galinhas , Coccidiose , Eimeria , Imunidade nas Mucosas , Interleucina-1beta , Doenças das Aves Domésticas , Vacinas Protozoárias , Animais , Coccidiose/imunologia , Coccidiose/veterinária , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Galinhas/imunologia , Eimeria/imunologia , Vacinas Protozoárias/imunologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/parasitologia , Doenças das Aves Domésticas/prevenção & controle , Imunização , Oocistos/imunologia , Microrganismos Geneticamente ModificadosRESUMO
Toxoplasma gondii is an obligate intracellular parasite capable of causing fatal infections in immunocompromised individuals and neonates. Examination of the phosphatidylserine (PtdSer) metabolism of T. gondii reveals that the parasite secretes a soluble form of PtdSer decarboxylase (TgPSD1), which preferentially decarboxylates liposomal PtdSer with an apparent K(m) of 67 µM. The specific enzyme activity increases by 3-fold during the replication of T. gondii, and soluble phosphatidylserine decarboxylase (PSD) accounts for â¼20% of the total PSD, prior to the parasite egress from the host cells. Extracellular T. gondii secreted â¼20% of its total PSD activity at 37 °C, and the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) inhibited the process by 50%. Cycloheximide, brefeldin A, ionic composition of the medium, and exogenous PtdSer did not modulate the enzyme secretion, which suggests a constitutive discharge of a presynthesized pool of PSD in axenic T. gondii. TgPSD1 consists of 968 amino acids with a 26-amino acid hydrophobic peptide at the N terminus and no predicted membrane domains. Parasites overexpressing TgPSD1-HA secreted 10-fold more activity compared with the parental strain. Exposure of apoptotic Jurkat cells to transgenic parasites demonstrated interfacial catalysis by secreted TgPSD1 that reduced host cell surface exposure of PtdSer. Immunolocalization experiments revealed that TgPSD1 resides in the dense granules of T. gondii and is also found in the parasitophorous vacuole of replicating parasites. Together, these findings demonstrate novel features of the parasite enzyme because a secreted, soluble, and interfacially active form of PSD has not been previously described for any organism.
Assuntos
Carboxiliases/genética , Carboxiliases/metabolismo , Fosfatidilserinas/metabolismo , Toxoplasma/enzimologia , Toxoplasmose/metabolismo , Toxoplasmose/microbiologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Cálcio/metabolismo , Carboxiliases/química , Catálise , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/parasitologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Células Jurkat , Metabolismo dos Lipídeos/fisiologia , Lipossomos/metabolismo , Dados de Sequência Molecular , Fagocitose/fisiologia , Filogenia , Estrutura Terciária de Proteína , Solubilidade , Temperatura , Vacúolos/enzimologiaRESUMO
The obligate intracellular apicomplexan parasites, e.g. Toxoplasma gondii and Plasmodium species, induce an IFNγ-driven induction of host indoleamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme of tryptophan catabolism in the kynurenine pathway. Induction of IDO1 supposedly depletes cellular levels of tryptophan in host cells, which is proposed to inhibit the in vitro growth of auxotrophic pathogens. In vivo function of IDO during infections, however, is not clear, let alone controversial. We show that Eimeria falciformis, an apicomplexan parasite infecting the mouse caecum, induces IDO1 in the epithelial cells of the organ, and the enzyme expression coincides with the parasite development. The absence or inhibition of IDO1/2 and of two downstream enzymes in infected animals is detrimental to the Eimeria growth. The reduced parasite yield is not due to a lack of an immunosuppressive effect of IDO1 in the parasitized IDO1(-/-) or inhibitor-treated mice because they did not show an accentuated Th1 and IFNγ response. Noticeably, the parasite development is entirely rescued by xanthurenic acid, a by-product of tryptophan catabolism inducing exflagellation in male gametes of Plasmodium in the mosquito mid-gut. Our data demonstrate a conceptual subversion of the host defense (IFNγ, IDO) by an intracellular pathogen for progression of its natural life cycle. Besides, we show utility of E. falciformis, a monoxenous parasite of a well appreciated host, i.e. mouse, to identify in vivo factors underlying the parasite-host interactions.
Assuntos
Coccidiose/metabolismo , Eimeria/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Células Th1/metabolismo , Triptofano/metabolismo , Animais , Coccidiose/genética , Coccidiose/imunologia , Culicidae/parasitologia , Eimeria/genética , Eimeria/imunologia , Inibidores Enzimáticos/farmacologia , Hipolipemiantes/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interferon gama/genética , Interferon gama/imunologia , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Células Th1/imunologia , Triptofano/genética , Triptofano/imunologia , Xanturenatos/farmacologiaRESUMO
The obligate intracellular and promiscuous protozoan parasite Toxoplasma gondii needs an extensive membrane biogenesis that must be satisfied irrespective of its host-cell milieu. We show that the synthesis of the major lipid in T. gondii, phosphatidylcholine (PtdCho), is initiated by a novel choline kinase (TgCK). Full-length (â¼70-kDa) TgCK displayed a low affinity for choline (K(m) â¼0.77 mM) and harbors a unique N-terminal hydrophobic peptide that is required for the formation of enzyme oligomers in the parasite cytosol but not for activity. Conditional mutagenesis of the TgCK gene in T. gondii attenuated the protein level by â¼60%, which was abolished in the off state of the mutant (Δtgck(i)). Unexpectedly, the mutant was not impaired in its growth and exhibited a normal PtdCho biogenesis. The parasite compensated for the loss of full-length TgCK by two potential 53- and 44-kDa isoforms expressed through a cryptic promoter identified within exon 1. TgCK-Exon1 alone was sufficient in driving the expression of GFP in E. coli. The presence of a cryptic promoter correlated with the persistent enzyme activity, PtdCho synthesis, and susceptibility of T. gondii to a choline analog, dimethylethanolamine. Quite notably, the mutant displayed a regular growth in the off state despite a 35% decline in PtdCho content and lipid synthesis, suggesting a compositional flexibility in the membranes of the parasite. The observed plasticity of gene expression and membrane biogenesis can ensure a faithful replication and adaptation of T. gondii in disparate host or nutrient environments.