scholarly journals Toxoplasma gondii LCAT Primarily Contributes to Tachyzoite Egress

mSphere ◽  
2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Aric J. Schultz ◽  
Vern B. Carruthers
Keyword(s):  
Catalytic Activity ◽  
Toxoplasma Gondii ◽  
Cholesterol Acyltransferase ◽  
Lytic Cycle ◽  
Human Pathogens ◽  
Content Type ◽  
Dense Granules ◽  
Lecithin Cholesterol Acyltransferase ◽  
Parasite Egress ◽  
Independent Mutant

ABSTRACT Egress is a crucial phase of the Toxoplasma gondii intracellular lytic cycle. This is a process that drives inflammation and is strongly associated with the pathogenesis observed during toxoplasmosis. Despite the link between this process and virulence, little is known about egress on a mechanistic or descriptive level. Previously published work has suggested that a phospholipase, lecithin-cholesterol acyltransferase (LCAT), secreted from the parasite’s dense granules contributes to parasite growth, virulence, and egress. Here we present evidence from several independent mutant parasite lines confirming a role for LCAT in efficient egress, although no defects in growth or virulence were apparent. We also show via genetic complementation that the catalytic activity of LCAT is required for its role in parasite egress. This work solidifies the contribution of LCAT to egress of T. gondii tachyzoites. IMPORTANCE Toxoplasma gondii is one of the most successful human pathogens, infecting an estimated 2.5 billion people across the globe. Pathogenesis seen during acute or reactivated toxoplasmosis has been closely tied to the parasite’s intracellular lytic life cycle, which culminates in an event called egress that results in the release of freshly replicated parasites from the infected host cell. Despite the highly destructive, cytolytic nature of this event and its downstream consequences, very little is known about how the parasite accomplishes this step. Previous work has suggested a role for a secreted phospholipase, LCAT, in Toxoplasma egress and roles in cell traversal and egress in the Plasmodium species orthologue. We confirm here that LCAT-deficient tachyzoites are unable to efficiently egress from infected monolayers, and we provide evidence that LCAT catalytic activity is required for its role in egress.

Effect of Ajuga iva on enzymes involved in the metabolism of cholesterol, in rat fed a cholesterol-enriched diet

2019 ◽  
Vol 50 (2) ◽  
pp. 303-313
Author(s):  
Sherazede Bouderbala ◽  
Malika Bouchenak
Keyword(s):  
Design Methodology ◽  
Cholesterol Metabolism ◽  
Cholesterol Acyltransferase ◽  
Cholesteryl Esters ◽  
Unesterified Cholesterol ◽  
Acyltransferase Activity ◽  
Content Type ◽  
Lecithin Cholesterol Acyltransferase ◽  
Male Wistar Rats ◽  
Adaptation Phase

Purpose This study aims to investigate the effect of Ajuga iva (Ai) on enzymes involved in the metabolism of cholesterol, in rat fed a cholesterol-enriched diet. Design/methodology/approach Male Wistar rats (n = 12), weighing 120 ± 5 g were fed on 1 per cent cholesterol-enriched diet [hypercholesterolemic (HC)] for 15 days (d15). After this adaptation phase, HC rats (total cholesterol = 6.5 ± 0.6 mmol/L) were divided into two groups fed the same diet and treated (Ai-HC) or not with (HC) with Ai for d15. Findings At day 15, in Ai-HC group compared to HC, serum triacylglycerol (TG) values were 1.4-fold lower (p = 0.002), whereas unesterified cholesterol (UC) contents were 1.8-fold higher (p = 0.0001). Serum phospholipids (PL) and cholesteryl esters (CE) contents and liver TG, UC, PL and CE values were not sensitive to Ai. TC/HDL-C and LDL-HDL1-C/HDL-C ratios were, respectively, 1.8- and 4-fold lower (p = 0.006 and p = 0.04). HDL2-C and HDL3-C amounts were enhanced by 40 and 74 per cent, respectively (p = 0.003 and p = 0.0001). HDL3-UC was 1.6-fold higher (p = 0.006); whereas PL contents were 1.4-fold lower (p = 0.003). HDL3-apo and HDL2-CE contents were similar between groups. A decreased of hydroxy-methyl-glutaryl-coenzyme A reductase and cholesterol 7α-hydroxylase activities (−44 and −25 per cent; p = 0.003 and p = 0.02, respectively) were noted. Lecithin: cholesterol acyltransferase activity was 1.5-fold higher (p = 0.001). Originality/value In HC rat, Ai is able to induce hypotriglyceridemia. However, it turns out that Ai may reduce cardiovascular risk by decreasing the reports of atherogenicity and modifying the activities of enzymes involved in the cholesterol metabolism.

scholarly journals Two Phosphoglucomutase Paralogs Facilitate Ionophore-Triggered Secretion of the Toxoplasma Micronemes

mSphere ◽  
2017 ◽  
Vol 2 (6) ◽  
Author(s):  
Sudeshna Saha ◽  
Bradley I. Coleman ◽  
Rashmi Dubey ◽  
Ira J. Blader ◽  
Marc-Jan Gubbels
Keyword(s):  
Toxoplasma Gondii ◽  
Phosphatidic Acid ◽  
Host Cell ◽  
Lytic Cycle ◽  
Ionophore A23187 ◽  
Host Cell Invasion ◽  
Content Type ◽  
Apicomplexan Parasites ◽  
Genetic Deletion

ABSTRACT Ca2+-dependent exocytosis is essential for the life cycle of apicomplexan parasites. Toxoplasma gondii harbors a phosphoglucomutase (PGM) ortholog, PRP1, previously associated with Ca2+-dependent microneme secretion. Here it is shown that genetic deletion of either PRP1, its PGM2 ortholog, or both genes is dispensable for the parasite’s lytic cycle, including host cell egress and invasion. Depletion of the proteins abrogated high Ca2+-mediated microneme secretion induced by the ionophore A23187; however, the constitutive and phosphatidic acid-mediated release remained unaffected. Secretion mediated by the former pathway is not essential for tachyzoite survival or acute in vivo infection in the mice. Paralogs of the widely prevalent phosphoglucomutase (PGM) protein called parafusin function in calcium (Ca2+)-mediated exocytosis across eukaryotes. In Toxoplasma gondii, the parafusin-related protein 1 (PRP1) has been associated with Ca2+-dependent microneme organelle secretion required for essential processes like host cell invasion and egress. Using reverse genetics, we observed PRP1 to be dispensable for completion of the lytic cycle, including host cell invasion and egress by the parasite. However, the absence of the gene affected increased microneme release triggered by A23187, a Ca2+ ionophore used to raise the cytoplasmic Ca2+ concentration mimicking the physiological role of Ca2+ during invasion and egress. The basal levels of constitutive microneme release in extracellular parasites and phosphatidic acid-triggered microneme secretion were unaffected in the mutant. The phenotype of the deletion mutant of the second PGM-encoding gene in Toxoplasma, PGM2, was similar to the phenotype of the PRP1 deletion mutant. Furthermore, the ability of the tachyzoites to induce acute infection in the mice remained normal in the absence of both PGM paralogs. Our data thus reveal that the microneme secretion upon high Ca2+ flux is facilitated by the Toxoplasma PGM paralogs, PRP1 and PGM2. However, this protein-mediated release is neither essential for lytic cycle completion nor for acute virulence of the parasite. IMPORTANCE Ca2+-dependent exocytosis is essential for the life cycle of apicomplexan parasites. Toxoplasma gondii harbors a phosphoglucomutase (PGM) ortholog, PRP1, previously associated with Ca2+-dependent microneme secretion. Here it is shown that genetic deletion of either PRP1, its PGM2 ortholog, or both genes is dispensable for the parasite’s lytic cycle, including host cell egress and invasion. Depletion of the proteins abrogated high Ca2+-mediated microneme secretion induced by the ionophore A23187; however, the constitutive and phosphatidic acid-mediated release remained unaffected. Secretion mediated by the former pathway is not essential for tachyzoite survival or acute in vivo infection in the mice.

scholarly journals Hydroxylamine and Carboxymethoxylamine Can Inhibit Toxoplasma gondii Growth through an Aspartate Aminotransferase-Independent Pathway

2020 ◽  
Vol 64 (3) ◽  
Author(s):  
Jixu Li ◽  
Huanping Guo ◽  
Eloiza May Galon ◽  
Yang Gao ◽  
Seung-Hun Lee ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Drug Targets ◽  
Protozoan Parasite ◽  
Cell Types ◽  
Lytic Cycle ◽  
Type I ◽  
Content Type ◽  
Mechanism Of Inhibition

ABSTRACT Toxoplasma gondii is an obligate intracellular protozoan parasite and a successful parasitic pathogen in diverse organisms and host cell types. Hydroxylamine (HYD) and carboxymethoxylamine (CAR) have been reported as inhibitors of aspartate aminotransferases (AATs) and interfere with the proliferation in Plasmodium falciparum. Therefore, AATs are suggested as drug targets against Plasmodium. The T. gondii genome encodes only one predicted AAT in both T. gondii type I strain RH and type II strain PLK. However, the effects of HYD and CAR, as well as their relationship with AAT, on T. gondii remain unclear. In this study, we found that HYD and CAR impaired the lytic cycle of T. gondii in vitro, including the inhibition of invasion or reinvasion, intracellular replication, and egress. Importantly, HYD and CAR could control acute toxoplasmosis in vivo. Further studies showed that HYD and CAR could inhibit the transamination activity of rTgAAT in vitro. However, our results confirmed that deficiency of AAT in both RH and PLK did not reduce the virulence in mice, although the growth ability of the parasites was affected in vitro. HYD and CAR could still inhibit the growth of AAT-deficient parasites. These findings indicated that HYD and CAR inhibition of T. gondii growth and control of toxoplasmosis can occur in an AAT-independent pathway. Overall, further studies focusing on the elucidation of the mechanism of inhibition are warranted. Our study hints at new substrates of HYD and CAR as potential drug targets to inhibit T. gondii growth.

scholarly journals The Vacuolar Zinc Transporter TgZnT Protects Toxoplasma gondii from Zinc Toxicity

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Nathan M. Chasen ◽  
Andrew J. Stasic ◽  
Beejan Asady ◽  
Isabelle Coppens ◽  
Silvia N. J. Moreno
Keyword(s):  
Toxoplasma Gondii ◽  
Metal Ion ◽  
Essential Element ◽  
Lytic Cycle ◽  
Zinc Toxicity ◽  
Content Type ◽  
Zinc Concentrations ◽  
High Concentrations ◽  
Yeast Mutants

ABSTRACT Zinc (Zn2+) is the most abundant biological metal ion aside from iron and is an essential element in numerous biological systems, acting as a cofactor for a large number of enzymes and regulatory proteins. Zn2+ must be tightly regulated, as both the deficiency and overabundance of intracellular free Zn2+ are harmful to cells. Zn2+ transporters (ZnTs) play important functions in cells by reducing intracellular Zn2+ levels by transporting the ion out of the cytoplasm. We characterized a Toxoplasma gondii gene (TgGT1_251630, TgZnT), which is annotated as the only ZnT family Zn2+ transporter in T. gondii. TgZnT localizes to novel vesicles that fuse with the plant-like vacuole (PLV), an endosome-like organelle. Mutant parasites lacking TgZnT exhibit reduced viability in in vitro assays. This phenotype was exacerbated by increasing zinc concentrations in the extracellular media and was rescued by media with reduced zinc. Heterologous expression of TgZnT in a Zn2+-sensitive Saccharomyces cerevisiae yeast strain partially restored growth in media with higher Zn2+ concentrations. These results suggest that TgZnT transports Zn2+ into the PLV and plays an important role in the Zn2+ tolerance of T. gondii extracellular tachyzoites. IMPORTANCE Toxoplasma gondii is an intracellular pathogen of human and animals. T. gondii pathogenesis is associated with its lytic cycle, which involves invasion, replication, egress out of the host cell, and invasion of a new one. T. gondii must be able to tolerate abrupt changes in the composition of the surrounding milieu as it progresses through its lytic cycle. We report the characterization of a Zn2+ transporter of T. gondii (TgZnT) that is important for parasite growth. TgZnT restored Zn2+ tolerance in yeast mutants that were unable to grow in media with high concentrations of Zn2+. We propose that TgZnT plays a role in Zn2+ homeostasis during the T. gondii lytic cycle.

scholarly journals Effects of PERK eIF2α Kinase Inhibitor against Toxoplasma gondii

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Leonardo Augusto ◽  
Jennifer Martynowicz ◽  
Kirk A. Staschke ◽  
Ronald C. Wek ◽  
William J. Sullivan
Keyword(s):  
Endoplasmic Reticulum ◽  
Toxoplasma Gondii ◽  
Er Stress ◽  
Lethal Dose ◽  
Lytic Cycle ◽  
Host Cells ◽  
Initiation Factor ◽  
Α Subunit ◽  
Content Type ◽  
Eif2α Kinase

ABSTRACT Toxoplasma gondii is an obligate intracellular parasite that has infected one-third of the population. Upon infection of warm-blooded vertebrates, the replicating form of the parasite (tachyzoite) converts into a latent form (bradyzoite) present in tissue cysts. During immune deficiency, bradyzoites can reconvert into tachyzoites and cause life-threatening toxoplasmosis. We previously reported that translational control through phosphorylation of the α subunit of T. gondii eukaryotic initiation factor 2 (eIF2α) (TgIF2α) is a critical component of the parasite stress response. Diverse stresses can induce the conversion of tachyzoites to bradyzoites, including those disrupting the parasite's endoplasmic reticulum (ER) (ER stress). Toxoplasma possesses four eIF2α kinases, one of which (TgIF2K-A) localizes to the parasite ER analogously to protein kinase R-like endoplasmic reticulum kinase (PERK), the eIF2α kinase that responds to ER stress in mammalian cells. Here, we investigated the effects of a PERK inhibitor (PERKi) on Toxoplasma. Our results show that the PERKi GSK2606414 blocks the enzymatic activity of TgIF2K-A and reduces TgIF2α phosphorylation specifically in response to ER stress. PERKi also significantly impeded multiple steps of the tachyzoite lytic cycle and sharply lowered the frequency of bradyzoite differentiation in vitro. Pretreatment of host cells with PERKi prior to infection did not affect parasite infectivity, and PERKi still impaired parasite replication in host cells lacking PERK. In mice, PERKi conferred modest protection from a lethal dose of Toxoplasma. Our findings represent the first pharmacological evidence supporting TgIF2K-A as an attractive new target for the treatment of toxoplasmosis.

scholarly journals Toxoplasma gondii Toxolysin 4 Contributes to Efficient Parasite Egress from Host Cells

mSphere ◽  
2021 ◽  
Author(s):  
My-Hang Huynh ◽  
Marijo S. Roiko ◽  
Angelica O. Gomes ◽  
Ellyn N. Schinke ◽  
Aric J. Schultz ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cells ◽  
Infected Host ◽  
Content Type ◽  
Parasite Egress

After replicating within infected host cells, the single-celled parasite Toxoplasma gondii must rupture out of such cells in a process termed egress. Although it is known that T. gondii egress is an active event that involves disruption of host-derived membranes surrounding the parasite, very few proteins that are released by the parasite are known to facilitate egress.

scholarly journals Molecular Characterization of Toxoplasma gondii Formin 3, an Actin Nucleator Dispensable for Tachyzoite Growth and Motility

2011 ◽  
Vol 11 (3) ◽  
pp. 343-352 ◽  
Author(s):  
Wassim Daher ◽  
Natacha Klages ◽  
Marie-France Carlier ◽  
Dominique Soldati-Favre
Keyword(s):  
Toxoplasma Gondii ◽  
Actin Polymerization ◽  
Life Stage ◽  
Gliding Motility ◽  
Lytic Cycle ◽  
Host Cells ◽  
Content Type ◽  
Parasite Motility ◽  
Actin Nucleator

ABSTRACT Toxoplasma gondii belongs to the phylum Apicomplexa, a group of obligate intracellular parasites that rely on gliding motility to enter host cells. Drugs interfering with the actin cytoskeleton block parasite motility, host cell invasion, and egress from infected cells. Myosin A, profilin, formin 1, formin 2, and actin-depolymerizing factor have all been implicated in parasite motility, yet little is known regarding the importance of actin polymerization and other myosins for the remaining steps of the parasite lytic cycle. Here we establish that T. gondii formin 3 (TgFRM3), a newly described formin homology 2 domain (FH2)-containing protein, binds to Toxoplasma actin and nucleates rabbit actin assembly in vitro . TgFRM3 expressed as a transgene exhibits a patchy localization at several distinct structures within the parasite. Disruption of the TgFRM3 gene by double homologous recombination in a ku80-ko strain reveals no vital function for tachyzoite propagation in vitro , which is consistent with its weak level of expression in this life stage. Conditional stabilization of truncated forms of TgFRM3 suggests that different regions of the molecule contribute to distinct localizations. Moreover, expression of TgFRM3 lacking the C-terminal domain severely affects parasite growth and replication. This work provides a first insight into how this specialized formin, restricted to the group of coccidia, completes its actin-nucleating activity.

scholarly journals Toxoplasma gondii excretion of glycolytic products is associated with acidification of the parasitophorous vacuole during parasite egress

2021 ◽  
Author(s):  
My-Hang Huynh ◽  
Vern B. Carruthers
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Indirect Evidence ◽  
Lytic Cycle ◽  
Vacuolar Acidification ◽  
Lactate And Pyruvate ◽  
Acidic Conditions ◽  
Parasite Egress

The Toxoplasma gondii lytic cycle is a repetition of host cell invasion, replication, egress, and re-invasion into the next host cell. While the molecular players involved in egress have been studied in greater detail in recent years, the signals and pathways for triggering egress from the host cell have not been fully elucidated. A perforin-like protein, PLP1, has been shown to be necessary for permeabilizing the parasitophorous vacuole (PV) membrane or exit from the host cell. In vitro studies indicated that PLP1 is most active in acidic conditions, and indirect evidence using superecliptic pHluorin indicated that the PV pH drops prior to parasite egress. Using ratiometric pHluorin, a GFP variant that responds to changes in pH with changes in its bimodal excitation spectrum peaks, allowed us to directly measure the pH in the PV prior to and during egress by live-imaging microscopy. A statistically significant change was observed in PV pH during egress in both wild-type RH and Δplp1 vacuoles compared to DMSO-treated vacuoles. Interestingly, if parasites are chemically paralyzed, a pH drop is still observed in RH but not in Δplp1 tachyzoites. This indicates that the pH drop is dependent on the presence of PLP1 or motility. Efforts to determine transporters, exchangers, or pumps that could contribute to the drop in PV pH identified two formate-nitrite transporters (FNTs). Auxin-induced conditional knockdown and knockouts of FNT1 and FNT2 reduced the levels of lactate and pyruvate released by the parasites and lead an abatement of vacuolar acidification. While additional transporters and molecules are undoubtedly involved, we provide evidence of a definitive reduction in vacuolar pH associated with induced and natural egress and characterize two transporters that contribute to the acidification.

scholarly journals A Glycosylphosphatidylinositol-Anchored Carbonic Anhydrase-Related Protein of Toxoplasma gondii Is Important for Rhoptry Biogenesis and Virulence

mSphere ◽  
2017 ◽  
Vol 2 (3) ◽  
Author(s):  
Nathan M. Chasen ◽  
Beejan Asady ◽  
Leandro Lemgruber ◽  
Rossiane C. Vommaro ◽  
Jessica C. Kissinger ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Carbonic Anhydrase ◽  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Lytic Cycle ◽  
Host Cells ◽  
Intracellular Pathogen ◽  
Related Protein ◽  
Content Type ◽  
Initial Interaction

ABSTRACT Toxoplasma gondii is an intracellular pathogen that infects humans and animals. The pathogenesis of T. gondii is linked to its lytic cycle, which starts when tachyzoites invade host cells and secrete proteins from specialized organelles. Once inside the host cell, the parasite creates a parasitophorous vacuole (PV) where it divides. Rhoptries are specialized secretory organelles that contain proteins, many of which are secreted during invasion. These proteins have important roles not only during the initial interaction between parasite and host but also in the formation of the PV and in the modification of the host cell. We report here the identification of a new T. gondii carbonic anhydrase-related protein (TgCA_RP), which localizes to rhoptries of mature tachyzoites. TgCA_RP is important for the morphology of rhoptries and for invasion and growth of parasites. TgCA_RP is also critical for parasite virulence. We propose that TgCA_RP plays a role in the biogenesis of rhoptries. Carbonic anhydrase-related proteins (CARPs) have previously been described as catalytically inactive proteins closely related to α-carbonic anhydrases (α-CAs). These CARPs are found in animals (both vertebrates and invertebrates) and viruses as either independent proteins or domains of other proteins. We report here the identification of a new CARP (TgCA_RP) in the unicellular organism Toxoplasma gondii that is related to the recently described η-class CA found in Plasmodium falciparum. TgCA_RP is posttranslationally modified at its C terminus with a glycosylphosphatidylinositol anchor that is important for its localization in intracellular tachyzoites. The protein localizes throughout the rhoptry bulbs of mature tachyzoites and to the outer membrane of nascent rhoptries in dividing tachyzoites, as demonstrated by immunofluorescence and immunoelectron microscopy using specific antibodies. T. gondii mutant tachyzoites lacking TgCA_RP display a growth and invasion phenotype in vitro and have atypical rhoptry morphology. The mutants also exhibit reduced virulence in a mouse model. Our results show that TgCA_RP plays an important role in the biogenesis of rhoptries. IMPORTANCE Toxoplasma gondii is an intracellular pathogen that infects humans and animals. The pathogenesis of T. gondii is linked to its lytic cycle, which starts when tachyzoites invade host cells and secrete proteins from specialized organelles. Once inside the host cell, the parasite creates a parasitophorous vacuole (PV) where it divides. Rhoptries are specialized secretory organelles that contain proteins, many of which are secreted during invasion. These proteins have important roles not only during the initial interaction between parasite and host but also in the formation of the PV and in the modification of the host cell. We report here the identification of a new T. gondii carbonic anhydrase-related protein (TgCA_RP), which localizes to rhoptries of mature tachyzoites. TgCA_RP is important for the morphology of rhoptries and for invasion and growth of parasites. TgCA_RP is also critical for parasite virulence. We propose that TgCA_RP plays a role in the biogenesis of rhoptries.

scholarly journals Pyruvate Homeostasis as a Determinant of Parasite Growth and Metabolic Plasticity inToxoplasma gondii

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Ningbo Xia ◽  
Shu Ye ◽  
Xiaohan Liang ◽  
Pu Chen ◽  
Yanqin Zhou ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Carbon Metabolism ◽  
Critical Role ◽  
Carbon Sources ◽  
Parasite Growth ◽  
Lytic Cycle ◽  
Current Understanding ◽  
Metabolic Flexibility ◽  
Lactate Dehydrogenase Activity ◽  
Content Type

ABSTRACTToxoplasma gondiiis a widespread intracellular pathogen infecting humans and a variety of animals. Previous studies have shown thatToxoplasmauses glucose and glutamine as the main carbon sources to support asexual reproduction, but neither nutrient is essential. Such metabolic flexibility may allow it to survive within diverse host cell types. Here, by focusing on the glycolytic enzyme pyruvate kinase (PYK) that converts phosphoenolpyruvate (PEP) into pyruvate, we found thatToxoplasmacan also utilize lactate and alanine. We show that catabolism of all indicated carbon sources converges at pyruvate, and maintaining a constant pyruvate supply is critical to parasite growth.Toxoplasmaexpresses two PYKs: PYK1 in the cytosol and PYK2 in the apicoplast (a chloroplast relict). Genetic deletion ofPYK2did not noticeably affect parasite growth and virulence, which contrasts with the current model of carbon metabolism in the apicoplast. On the other hand,PYK1was refractory to disruption. Conditional depletion of PYK1 resulted in global alteration of carbon metabolism, amylopectin accumulation, and reduced cellular ATP, leading to severe growth impairment. Notably, the attenuated growth of the PYK1-depleted mutant was partially rescued by lactate or alanine supplementation, and rescue by lactate required lactate dehydrogenase activity to convert it to pyruvate. Moreover, depletion of PYK1 in conjunction with PYK2 ablation led to accentuated loss of apicoplasts and complete growth arrest. Together, our results underline a critical role of pyruvate homeostasis in determining the metabolic flexibility and apicoplast maintenance, and they significantly extend our current understanding of carbon metabolism inT. gondii.IMPORTANCEToxoplasma gondiiinfects almost all warm-blooded animals, and metabolic flexibility is deemed critical for its successful parasitism in diverse hosts. Glucose and glutamine are the major carbon sources to support parasite growth. In this study, we found thatToxoplasmais also competent in utilizing lactate and alanine and, thus, exhibits exceptional metabolic versatility. Notably, all these nutrients need to be converted to pyruvate to fuel the lytic cycle, and achieving a continued pyruvate supply is vital to parasite survival and metabolic flexibility. Although pyruvate can be generated by two distinct pyruvate kinases, located in cytosol and apicoplast, respectively, the cytosolic enzyme is the main source of subcellular pyruvate, and cooperative usage of pyruvate among multiple organelles is critical for parasite growth and virulence. These findings expand our current understanding of carbon metabolism inToxoplasma gondiiand related parasites while providing a basis for designing novel antiparasitic interventions.

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