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1.
J Lipid Res ; 65(10): 100645, 2024 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-39306040

RESUMEN

Apicomplexa comprise important pathogenic parasitic protists that heavily depend on lipid acquisition to survive within their human host cells. Lipid synthesis relies on the incorporation of an essential combination of fatty acids (FAs) either generated by a metabolically adaptable de novo synthesis in the parasite or by scavenging from the host cell. The incorporation of FAs into membrane lipids depends on their obligate metabolic activation by specific enzyme groups, acyl-CoA synthetases (ACSs). Each ACS has its own specificity, so it can fulfill specific metabolic functions. Whilst such functionalities have been well studied in other eukaryotic models, their roles and importance in Apicomplexa are currently very limited, especially for Toxoplasma gondii. Here, we report the identification of seven putative ACSs encoded by the genome of T. gondii (TgACS), which localize to different sub-cellular compartments of the parasite, suggesting exclusive functions. We show that the perinuclear/cytoplasmic TgACS3 regulates the replication and growth of Toxoplasma tachyzoites. Conditional disruption of TgACS3 shows that the enzyme is required for parasite propagation and survival, especially under high host nutrient content. Lipidomic analysis of parasites lacking TgACS3 reveals its role in the activation of host-derived FAs that are used for i) parasite membrane phospholipid and ii) storage triacylglycerol (TAG) syntheses, allowing proper membrane biogenesis of parasite progenies. Altogether, our results reveal the role of TgACS3 as the bulk FA activator for membrane biogenesis allowing intracellular division and survival in T. gondii tachyzoites, further pointing to the importance of ACS and FA metabolism for the parasite.

2.
EMBO Rep ; 23(2): e54022, 2022 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-34866326

RESUMEN

PfCDPK7 is an atypical member of the calcium-dependent protein kinase (CDPK) family and is crucial for the development of Plasmodium falciparum. However, the mechanisms whereby PfCDPK7 regulates parasite development remain unknown. Here, we perform quantitative phosphoproteomics and phospholipid analysis and find that PfCDPK7 promotes phosphatidylcholine (PC) synthesis by regulating two key enzymes involved in PC synthesis, phosphoethanolamine-N-methyltransferase (PMT) and ethanolamine kinase (EK). In the absence of PfCDPK7, both enzymes are hypophosphorylated and PMT is degraded. We further find that PfCDPK7 interacts with 4'-phosphorylated phosphoinositides (PIPs) generated by PI4-kinase. Inhibition of PI4K activity disrupts the vesicular localization PfCDPK7. P. falciparum PI4-kinase, PfPI4K is a prominent drug target and one of its inhibitors, MMV39048, has reached Phase I clinical trials. Using this inhibitor, we demonstrate that PfPI4K controls phospholipid biosynthesis and may act in part by regulating PfCDPK7 localization and activity. These studies not only unravel a signaling pathway involving PfPI4K/4'-PIPs and PfCDPK7 but also provide novel insights into the mechanism of action of a promising series of candidate anti-malarial drugs.


Asunto(s)
Malaria Falciparum , Plasmodium falciparum , Humanos , Fosfolípidos/metabolismo , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Transducción de Señal
3.
J Biol Chem ; 298(8): 102243, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35810787

RESUMEN

Like many other apicomplexan parasites, Toxoplasma gondii contains a plastid harboring key metabolic pathways, including the sulfur utilization factor (SUF) pathway that is involved in the biosynthesis of iron-sulfur clusters. These cofactors are crucial for a variety of proteins involved in important metabolic reactions, potentially including plastidic pathways for the synthesis of isoprenoid and fatty acids. It was shown previously that impairing the NFS2 cysteine desulfurase, involved in the first step of the SUF pathway, leads to an irreversible killing of intracellular parasites. However, the metabolic impact of disrupting the pathway remained unexplored. Here, we generated another mutant of this pathway, deficient in the SUFC ATPase, and investigated in details the phenotypic consequences of TgNFS2 and TgSUFC depletion on the parasites. Our analysis confirms that Toxoplasma SUF mutants are severely and irreversibly impacted in division and membrane homeostasis, and suggests a defect in apicoplast-generated fatty acids. However, we show that increased scavenging from the host or supplementation with exogenous fatty acids do not fully restore parasite growth, suggesting that this is not the primary cause for the demise of the parasites and that other important cellular functions were affected. For instance, we also show that the SUF pathway is key for generating the isoprenoid-derived precursors necessary for the proper targeting of GPI-anchored proteins and for parasite motility. Thus, we conclude plastid-generated iron-sulfur clusters support the functions of proteins involved in several vital downstream cellular pathways, which implies the SUF machinery may be explored for new potential anti-Toxoplasma targets.


Asunto(s)
Apicoplastos , Proteínas Hierro-Azufre , Proteínas Protozoarias , Toxoplasma , Apicoplastos/genética , Apicoplastos/metabolismo , Ácidos Grasos/metabolismo , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Plastidios/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Terpenos/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismo
4.
PLoS Pathog ; 17(2): e1009325, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33635921

RESUMEN

Apicomplexan parasites are causative agents of major human diseases. Calcium Dependent Protein Kinases (CDPKs) are crucial components for the intracellular development of apicomplexan parasites and are thus considered attractive drug targets. CDPK7 is an atypical member of this family, which initial characterization suggested to be critical for intracellular development of both Apicomplexa Plasmodium falciparum and Toxoplasma gondii. However, the mechanisms via which it regulates parasite replication have remained unknown. We performed quantitative phosphoproteomics of T. gondii lacking TgCDPK7 to identify its parasitic targets. Our analysis lead to the identification of several putative TgCDPK7 substrates implicated in critical processes like phospholipid (PL) synthesis and vesicular trafficking. Strikingly, phosphorylation of TgRab11a via TgCDPK7 was critical for parasite intracellular development and protein trafficking. Lipidomic analysis combined with biochemical and cellular studies confirmed that TgCDPK7 regulates phosphatidylethanolamine (PE) levels in T. gondii. These studies provide novel insights into the regulation of these processes that are critical for parasite development by TgCDPK7.


Asunto(s)
Lipogénesis , Fosfatidiletanolaminas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/enzimología , Toxoplasmosis/metabolismo , Vesículas Transportadoras/metabolismo , Transporte Biológico , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/parasitología , Humanos , Fosforilación , Proteínas Quinasas/genética , Proteínas Protozoarias/genética , Toxoplasma/crecimiento & desarrollo , Toxoplasmosis/parasitología
5.
Int J Mol Sci ; 24(6)2023 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-36983080

RESUMEN

Several studies have linked bad prognoses of acute myeloid leukemia (AML) to the ability of leukemic cells to reprogram their metabolism and, in particular, their lipid metabolism. In this context, we performed "in-depth" characterization of fatty acids (FAs) and lipid species in leukemic cell lines and in plasma from AML patients. We firstly showed that leukemic cell lines harbored significant differences in their lipid profiles at steady state, and that under nutrient stress, they developed common mechanisms of protection that led to variation in the same lipid species; this highlights that the remodeling of lipid species is a major and shared mechanism of adaptation to stress in leukemic cells. We also showed that sensitivity to etomoxir, which blocks fatty acid oxidation (FAO), was dependent on the initial lipid profile of cell lines, suggesting that only a particular "lipidic phenotype" is sensitive to the drug targeting of FAO. We then showed that the lipid profiles of plasma samples from AML patients were significantly correlated with the prognosis of patients. In particular, we highlighted the impact of phosphocholine and phosphatidyl-choline metabolism on patients' survival. In conclusion, our data show that balance between lipid species is a phenotypic marker of the diversity of leukemic cells that significantly influences their proliferation and resistance to stress, and thereby, the prognosis of AML patients.


Asunto(s)
Leucemia Mieloide Aguda , Metabolismo de los Lípidos , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Ácidos Grasos/metabolismo , Sistemas de Liberación de Medicamentos
6.
BMC Biol ; 19(1): 159, 2021 08 11.
Artículo en Inglés | MEDLINE | ID: mdl-34380472

RESUMEN

BACKGROUND: Plasmodium falciparum is the pathogen responsible for the most devastating form of human malaria. As it replicates asexually in the erythrocytes of its human host, the parasite feeds on haemoglobin uptaken from these cells. Heme, a toxic by-product of haemoglobin utilization by the parasite, is neutralized into inert hemozoin in the food vacuole of the parasite. Lipid homeostasis and phospholipid metabolism are crucial for this process, as well as for the parasite's survival and propagation within the host. P. falciparum harbours a uniquely large family of phospholipases, which are suggested to play key roles in lipid metabolism and utilization. RESULTS: Here, we show that one of the parasite phospholipase (P. falciparum lysophospholipase, PfLPL1) plays an essential role in lipid homeostasis linked with the haemoglobin degradation and heme conversion pathway. Fluorescence tagging showed that the PfLPL1 in infected blood cells localizes to dynamic vesicular structures that traffic from the host-parasite interface at the parasite periphery, through the cytosol, to get incorporated into a large vesicular lipid rich body next to the food-vacuole. PfLPL1 is shown to harbour enzymatic activity to catabolize phospholipids, and its transient downregulation in the parasite caused a significant reduction of neutral lipids in the food vacuole-associated lipid bodies. This hindered the conversion of heme, originating from host haemoglobin, into the hemozoin, and disrupted the parasite development cycle and parasite growth. Detailed lipidomic analyses of inducible knock-down parasites deciphered the functional role of PfLPL1 in generation of neutral lipid through recycling of phospholipids. Further, exogenous fatty-acids were able to complement downregulation of PfLPL1 to rescue the parasite growth as well as restore hemozoin levels. CONCLUSIONS: We found that the transient downregulation of PfLPL1 in the parasite disrupted lipid homeostasis and caused a reduction in neutral lipids essentially required for heme to hemozoin conversion. Our study suggests a crucial link between phospholipid catabolism and generation of neutral lipids (TAGs) with the host haemoglobin degradation pathway.


Asunto(s)
Malaria Falciparum , Plasmodium falciparum , Eritrocitos , Hemo , Hemoproteínas , Humanos , Fosfolipasas , Fosfolípidos
7.
J Biol Chem ; 295(18): 6108-6119, 2020 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-32217691

RESUMEN

Pathogenic bacteria of the genera Mycobacterium and Corynebacterium cause severe human diseases such as tuberculosis (Mycobacterium tuberculosis) and diphtheria (Corynebacterium diphtheriae). The cells of these species are surrounded by protective cell walls rich in long-chain mycolic acids. These fatty acids are conjugated to the disaccharide trehalose on the cytoplasmic side of the bacterial cell membrane. They are then transported across the membrane to the periplasm where they act as donors for other reactions. We have previously shown that transient acetylation of the glycolipid trehalose monohydroxycorynomycolate (hTMCM) enables its efficient transport to the periplasm in Corynebacterium glutamicum and that acetylation is mediated by the membrane protein TmaT. Here, we show that a putative methyltransferase, encoded at the same genetic locus as TmaT, is also required for optimal hTMCM transport. Deletion of the C. glutamicum gene NCgl2764 (Rv0224c in M. tuberculosis) abolished acetyltrehalose monocorynomycolate (AcTMCM) synthesis, leading to accumulation of hTMCM in the inner membrane and delaying its conversion to trehalose dihydroxycorynomycolate (h2TDCM). Complementation with NCgl2764 normalized turnover of hTMCM to h2TDCM. In contrast, complementation with NCgl2764 derivatives mutated at residues essential for methyltransferase activity failed to rectify the defect, suggesting that NCgl2764/Rv0224c encodes a methyltransferase, designated here as MtrP. Comprehensive analyses of the individual mtrP and tmaT mutants and of a double mutant revealed strikingly similar changes across several lipid classes compared with WT bacteria. These findings indicate that both MtrP and TmaT have nonredundant roles in regulating AcTMCM synthesis, revealing additional complexity in the regulation of trehalose mycolate transport in the Corynebacterineae.


Asunto(s)
Membrana Celular/metabolismo , Corynebacterium glutamicum/citología , Corynebacterium glutamicum/enzimología , Metiltransferasas/metabolismo , Ácidos Micólicos/química , Trehalosa/química , Trehalosa/metabolismo , Transporte Biológico , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Metiltransferasas/genética , Mutación , Mycobacterium tuberculosis/genética , Homología de Secuencia de Ácido Nucleico
8.
Cell Microbiol ; 22(3): e13146, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31734953

RESUMEN

Patatin-like phospholipases (PNPLAs) are highly conserved enzymes of prokaryotic and eukaryotic organisms with major roles in lipid homeostasis. The genome of the malaria parasite Plasmodium falciparum encodes four putative PNPLAs with predicted functions during phospholipid degradation. We here investigated the role of one of the plasmodial PNPLAs, a putative PLA2 termed PNPLA1, during blood stage replication and gametocyte development. PNPLA1 is present in the asexual and sexual blood stages and here localizes to the cytoplasm. PNPLA1-deficiency due to gene disruption or conditional gene-knockdown had no effect on intraerythrocytic growth, gametocyte development and gametogenesis. However, parasites lacking PNPLA1 were impaired in gametocyte induction, while PNPLA1 overexpression promotes gametocyte formation. The loss of PNPLA1 further leads to transcriptional down-regulation of genes related to gametocytogenesis, including the gene encoding the sexual commitment regulator AP2-G. Additionally, lipidomics of PNPLA1-deficient asexual blood stage parasites revealed overall increased levels of major phospholipids, including phosphatidylcholine (PC), which is a substrate of PLA2 . PC synthesis is known to be pivotal for erythrocytic replication, while the reduced availability of PC precursors drives the parasite into gametocytogenesis; we thus hypothesize that the higher PC levels due to PNPLA1-deficiency prevent the blood stage parasites from entering the sexual pathway.


Asunto(s)
Fosfolipasas/fisiología , Plasmodium falciparum/fisiología , Proteínas Protozoarias/fisiología , Animales , Citoplasma/genética , Citoplasma/metabolismo , Femenino , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genoma de Protozoos , Estadios del Ciclo de Vida , Metabolismo de los Lípidos , Ratones , Fosfolipasas/genética , Plasmodium falciparum/enzimología , Proteínas Protozoarias/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
9.
J Biol Chem ; 294(22): 8959-8972, 2019 05 31.
Artículo en Inglés | MEDLINE | ID: mdl-30992368

RESUMEN

Protozoan parasites of the phylum Apicomplexa actively move through tissue to initiate and perpetuate infection. The regulation of parasite motility relies on cyclic nucleotide-dependent kinases, but how these kinases are activated remains unknown. Here, using an array of biochemical and cell biology approaches, we show that the apicomplexan parasite Toxoplasma gondii expresses a large guanylate cyclase (TgGC) protein, which contains several upstream ATPase transporter-like domains. We show that TgGC has a dynamic localization, being concentrated at the apical tip in extracellular parasites, which then relocates to a more cytosolic distribution during intracellular replication. Conditional TgGC knockdown revealed that this protein is essential for acute-stage tachyzoite growth, as TgGC-deficient parasites were defective in motility, host cell attachment, invasion, and subsequent host cell egress. We show that TgGC is critical for a rapid rise in cytosolic [Ca2+] and for secretion of microneme organelles upon stimulation with a cGMP agonist, but these deficiencies can be bypassed by direct activation of signaling by a Ca2+ ionophore. Furthermore, we found that TgGC is required for transducing changes in extracellular pH and [K+] to activate cytosolic [Ca2+] flux. Together, the results of our work implicate TgGC as a putative signal transducer that activates Ca2+ signaling and motility in Toxoplasma.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Señalización del Calcio , Guanilato Ciclasa/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Adenosina Trifosfatasas/genética , Calcio/metabolismo , Ionóforos de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , GMP Cíclico/metabolismo , Citosol/metabolismo , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/genética , Concentración de Iones de Hidrógeno , Oligonucleótidos Antisentido/metabolismo , Potasio/metabolismo , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genética , Pirazoles/farmacología , Pirimidinonas/farmacología , Toxoplasma/crecimiento & desarrollo
10.
J Lipid Res ; 59(6): 994-1004, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29678960

RESUMEN

Apicomplexan parasites are pathogens responsible for major human diseases such as toxoplasmosis caused by Toxoplasma gondii and malaria caused by Plasmodium spp. Throughout their intracellular division cycle, the parasites require vast and specific amounts of lipids to divide and survive. This demand for lipids relies on a fine balance between de novo synthesized lipids and scavenged lipids from the host. Acetyl-CoA is a major and central precursor for many metabolic pathways, especially for lipid biosynthesis. T. gondii possesses a single cytosolic acetyl-CoA synthetase (TgACS). Its role in the parasite lipid synthesis is unclear. Here, we generated an inducible TgACS KO parasite line and confirmed the cytosolic localization of the protein. We conducted 13C-stable isotope labeling combined with mass spectrometry-based lipidomic analyses to unravel its putative role in the parasite lipid synthesis pathway. We show that its disruption has a minor effect on the global FA composition due to the metabolic changes induced to compensate for its loss. However, we could demonstrate that TgACS is involved in providing acetyl-CoA for the essential fatty elongation pathway to generate FAs used for membrane biogenesis. This work provides novel metabolic insight to decipher the complex lipid synthesis in T. gondii.


Asunto(s)
Acetato CoA Ligasa/metabolismo , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Estadios del Ciclo de Vida , Toxoplasma/enzimología , Toxoplasma/crecimiento & desarrollo , Acetato CoA Ligasa/química , Secuencia de Aminoácidos , Citosol/metabolismo , Ácidos Grasos/biosíntesis , Modelos Moleculares , Nutrientes/metabolismo , Conformación Proteica , Toxoplasma/metabolismo
11.
J Biol Chem ; 292(12): 4976-4986, 2017 03 24.
Artículo en Inglés | MEDLINE | ID: mdl-28167532

RESUMEN

Mycobacterium tuberculosis and related Corynebacterineae synthesize a family of lipomannans (LM) and lipoarabinomannans (LAM) that are abundant components of the multilaminate cell wall and essential virulence factors in pathogenic species. Here we describe a new membrane protein, highly conserved in all Corynebacterineae, that is required for synthesis of full-length LM and LAM. Deletion of the Corynebacterium glutamicum NCgl2760 gene resulted in a complete loss of mature LM/LAM and the appearance of a truncated LM (t-LM). Complementation of the mutant with the NCgl2760 gene fully restored LM/LAM synthesis. Structural studies, including monosaccharide analysis, methylation linkage analysis, and mass spectrometry of native LM species, indicated that the ΔNCgl2760 t-LM comprised a series of short LM species (8-27 residues long) containing an α1-6-linked mannose backbone with greatly reduced α1-2-mannose side chains and no arabinose caps. The structure of the ΔNCgl2760 t-LM was similar to that of the t-LM produced by a C. glutamicum mutant lacking the mptA gene, encoding a membrane α1-6-mannosyltransferase involved in extending the α1-6-mannan backbone of LM intermediates. Interestingly, NCgl2760 lacks any motifs or homology to other proteins of known function. Attempts to delete the NCgl2760 orthologue in Mycobacterium smegmatis were unsuccessful, consistent with previous studies indicating that the M. tuberculosis orthologue, Rv0227c, is an essential gene. Together, these data suggest that NCgl2760/Rv0227c plays a critical role in the elongation of the mannan backbone of mycobacterial and corynebacterial LM, further highlighting the complexity of lipoglycan pathways of Corynebacterineae.


Asunto(s)
Proteínas Bacterianas/metabolismo , Corynebacterium glutamicum/metabolismo , Lipopolisacáridos/metabolismo , Proteínas Bacterianas/genética , Vías Biosintéticas , Pared Celular/genética , Pared Celular/metabolismo , Corynebacterium glutamicum/genética , Eliminación de Gen
12.
Mol Microbiol ; 105(1): 158-174, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28419631

RESUMEN

Patatin-like phospholipases are involved in numerous cellular functions, including lipid metabolism and membranes remodeling. The patatin-like catalytic domain, whose phospholipase activity relies on a serine-aspartate dyad and an anion binding box, is widely spread among prokaryotes and eukaryotes. We describe TgPL2, a novel patatin-like phospholipase domain-containing protein from the parasitic protist Toxoplasma gondii. TgPL2 is a large protein, in which the key motifs for enzymatic activity are conserved in the patatin-like domain. Using immunofluorescence assays and immunoelectron microscopy analysis, we have shown that TgPL2 localizes to the apicoplast, a non-photosynthetic plastid found in most apicomplexan parasites. This plastid hosts several important biosynthetic pathways, which makes it an attractive organelle for identifying new potential drug targets. We thus addressed TgPL2 function by generating a conditional knockdown mutant and demonstrated it has an essential contribution for maintaining the integrity of the plastid. In absence of TgPL2, the organelle is rapidly lost and remaining apicoplasts appear enlarged, with an abnormal accumulation of membranous structures, suggesting a defect in lipids homeostasis. More precisely, analyses of lipid content upon TgPL2 depletion suggest this protein is important for maintaining levels of apicoplast-generated fatty acids, and also regulating phosphatidylcholine and lysophosphatidylcholine levels in the parasite.


Asunto(s)
Apicoplastos/metabolismo , Fosfolipasas/metabolismo , Toxoplasma/metabolismo , Secuencia de Aminoácidos , Animales , Apicoplastos/genética , Secuencia de Bases , Dominio Catalítico , Ácidos Grasos/metabolismo , Homeostasis , Metabolismo de los Lípidos/fisiología , Lípidos , Parásitos , Plastidios/metabolismo , Dominios Proteicos , Proteínas Protozoarias/metabolismo
13.
PLoS Pathog ; 12(8): e1005765, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27490259

RESUMEN

Most apicomplexan parasites possess a non-photosynthetic plastid (the apicoplast), which harbors enzymes for a number of metabolic pathways, including a prokaryotic type II fatty acid synthesis (FASII) pathway. In Toxoplasma gondii, the causative agent of toxoplasmosis, the FASII pathway is essential for parasite growth and infectivity. However, little is known about the fate of fatty acids synthesized by FASII. In this study, we have investigated the function of a plant-like glycerol 3-phosphate acyltransferase (TgATS1) that localizes to the T. gondii apicoplast. Knock-down of TgATS1 resulted in significantly reduced incorporation of FASII-synthesized fatty acids into phosphatidic acid and downstream phospholipids and a severe defect in intracellular parasite replication and survival. Lipidomic analysis demonstrated that lipid precursors are made in, and exported from, the apicoplast for de novo biosynthesis of bulk phospholipids. This study reveals that the apicoplast-located FASII and ATS1, which are primarily used to generate plastid galactolipids in plants and algae, instead generate bulk phospholipids for membrane biogenesis in T. gondii.


Asunto(s)
Apicoplastos/enzimología , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Fosfolípidos/biosíntesis , Proteínas Protozoarias/biosíntesis , Toxoplasma/metabolismo , Secuencia de Aminoácidos , Cromatografía Liquida , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Lisofosfolípidos/biosíntesis , Espectrometría de Masas , Microscopía Electrónica de Transmisión , Modelos Moleculares , Filogenia , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/química
14.
Plant Physiol ; 170(3): 1300-14, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26620528

RESUMEN

The biogenesis of photosynthetic membranes relies on galactoglycerolipids, which are synthesized via pathways that are dispatched over several cell compartments. This membrane biogenesis requires both trafficking of lipid intermediates and a tight homeostatic regulation. In this work, we address the role of ALA10 (for aminophospholipid ATPase), a P4-type ATPase, in a process counteracting the monogalactosyldiacylglycerol (MGDG) shortage in Arabidopsis (Arabidopsis thaliana) leaves. ALA10 can interact with protein partners, ALIS1 (for ALA-interacting subunit1) or ALIS5, leading to differential endomembrane localizations of the interacting proteins, close to the plasma membrane with ALIS1 or to chloroplasts with ALIS5. ALA10 interacts also with FATTY ACID DESATURASE2 (FAD2), and modification of ALA10 expression affects phosphatidylcholine (PC) fatty acyl desaturation by disturbing the balance between FAD2 and FAD3 activities. Modulation of ALA10 expression downstream impacts the fatty acyl composition of chloroplast PC. ALA10 expression also enhances leaf growth and improves the MGDG-PC ratio, possibly through MGDG SYNTHASE1 (MGD1) activation by phosphatidic acid. The positive effect of ALA10 on leaf development is significant in conditions such as upon treatment of plants with Galvestine-1, an inhibitor of MGDG synthases, or when plants are grown at chilling temperature.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácido Graso Desaturasas/metabolismo , Fosfatidilcolinas/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Cloroplastos/metabolismo , Retículo Endoplásmico/metabolismo , Galactolípidos/metabolismo , Perfilación de la Expresión Génica , Metabolismo de los Lípidos , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente
15.
Proc Natl Acad Sci U S A ; 110(18): 7506-11, 2013 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-23589867

RESUMEN

The human malaria parasite Plasmodium falciparum harbors a relict, nonphotosynthetic plastid of algal origin termed the apicoplast. Although considerable progress has been made in defining the metabolic functions of the apicoplast, information on the composition and biogenesis of the four delimiting membranes of this organelle is limited. Here, we report an efficient method for preparing highly purified apicoplasts from red blood cell parasite stages and the comprehensive lipidomic analysis of this organelle. Apicoplasts were prepared from transgenic parasites expressing an epitope-tagged triosephosphate transporter and immunopurified on magnetic beads. Gas and liquid chromatography MS analyses of isolated apicoplast lipids indicated significant differences compared with total parasite lipids. In particular, apicoplasts were highly enriched in phosphatidylinositol, consistent with a suggested role for phosphoinositides in targeting membrane vesicles to apicoplasts. Apicoplast phosphatidylinositol and other phospholipids were also enriched in saturated fatty acids, which could reflect limited acyl exchange with other membrane phospholipids and/or a requirement for specific physical properties. Lipids atypical for plastids (sphingomyelins, ceramides, and cholesterol) were detected in apicoplasts. The presence of cholesterol in apicoplast membranes was supported by filipin staining of isolated apicoplasts. Galactoglycerolipids, dominant in plant and algal plastids, were not detected in P. falciparum apicoplasts, suggesting that these glycolipids are a hallmark of photosynthetic plastids and were lost when these organisms assumed a parasitic lifestyle. Apicoplasts thus contain an atypical melange of lipids scavenged from the human host alongside lipids remodeled by the parasite cytoplasm, and stable isotope labeling shows some apicoplast lipids are generated de novo by the organelle itself.


Asunto(s)
Lípidos/química , Malaria Falciparum/parasitología , Plasmodium falciparum/metabolismo , Plastidios/química , Colesterol/metabolismo , Cromatografía Liquida , Ácidos Grasos/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Humanos , Metabolismo de los Lípidos , Plasmodium falciparum/ultraestructura , Plastidios/ultraestructura
16.
Methods Mol Biol ; 2776: 43-62, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38502497

RESUMEN

Chloroplasts are essential organelles that are responsible for photosynthesis in a wide range of organisms that have colonized all biotopes on Earth such as plants and unicellular algae. Interestingly, a secondary endosymbiotic event of a red algal ancestor gave rise to a group of organisms that have adopted an obligate parasitic lifestyle named Apicomplexa parasites. Apicomplexa parasites are some of the most widespread and poorly controlled pathogens in the world. These infectious agents are responsible for major human diseases such as toxoplasmosis, caused by Toxoplasma gondii, and malaria, caused by Plasmodium spp. Most of these parasites harbor this relict plastid named the apicoplast, which is essential for parasite survival. The apicoplast has lost photosynthetic capacities but is metabolically similar to plant and algal chloroplasts. The apicoplast is considered a novel and important drug target against Apicomplexa parasites. This chapter focuses on the apicoplast of apicomplexa parasites, its maintenance, and its metabolic pathways.


Asunto(s)
Apicoplastos , Parásitos , Plasmodium , Toxoplasma , Animales , Humanos , Apicoplastos/genética , Apicoplastos/metabolismo , Simbiosis , Toxoplasma/genética , Toxoplasma/metabolismo
17.
Methods Mol Biol ; 2776: 197-204, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38502506

RESUMEN

Apicomplexan parasites are unicellular eukaryotes responsible for major human diseases such as malaria and toxoplasmosis, which cause massive social and economic burden. Toxoplasmosis, caused by Toxoplasma gondii, is a global chronic infectious disease affecting ~1/3 of the world population and is a major threat for any immunocompromised patient. To date, there is no efficient vaccine against these parasites and existing treatments are threatened by rapid emergence of parasite resistance. Throughout their life cycle, Apicomplexa require large amount of nutrients, especially lipids for propagation and survival. Understanding lipid acquisition is key to decipher host-parasite metabolic interactions. Parasite membrane biogenesis relies on a combination of (a) host lipid scavenging, (b) de novo lipid synthesis in the parasite, and (c) fluxes of lipids between host and parasite and within. We recently uncovered that parasite need to store the host-scavenged lipids to avoid their toxic accumulation and to mobilize them for division. How can parasites orchestrate the many lipids fluxes essential for survival? Here, we developed metabolomics approaches coupled to stable isotope labelling to track, monitor, and quantify fatty acid and lipids fluxes between the parasite, its human host cell, and its extracellular environment to unravel the complex lipid fluxes in any physiological environment the parasite could meet.


Asunto(s)
Parásitos , Toxoplasma , Toxoplasmosis , Animales , Humanos , Parásitos/metabolismo , Plastidios/metabolismo , Ácidos Grasos/metabolismo , Toxoplasma/metabolismo , Toxoplasmosis/metabolismo , Proteínas Protozoarias/metabolismo
18.
J Immunother Cancer ; 12(9)2024 Sep 24.
Artículo en Inglés | MEDLINE | ID: mdl-39317456

RESUMEN

BACKGROUND: Tumor-associated macrophages participate in the complex network of support that favors tumor growth. Among the various strategies that have been developed to target these cells, the blockade of the colony-stimulating factor 1 receptor (CSF-1R) receptor is one of the most promising ones. Here, we characterize the resulting state of human macrophages exposed to a CSF-1R kinase inhibitor. METHODS: Using RNA sequencing and metabolomics approach, we characterize the reprogramming of human monocyte-derived macrophages under CSF-1R targeting. RESULTS: We find that CSF-1R receptor inhibition in human macrophages is able to impair cholesterol synthesis, fatty acid metabolism and hypoxia-driven expression of dihydropyrimidine dehydrogenase, an enzyme responsible for the 5-fluorouracil macrophage-mediated chemoresistance. We show that this inhibition of the CSF-1R receptor leads to a downregulation of the expression of sterol regulatory element-binding protein 2, a transcription factor that controls cholesterol and fatty acid synthesis. We also show that the inhibition of extracellular signal-regulated kinase 1/2 phosphorylation resulting from targeting the CSF-1R receptor destabilizes the expression of hypoxic induced factor 2 alpha in hypoxia resulting in the downregulation of dihydropyrimidine dehydrogenase expression restoring the sensitivity to 5-fluorouracil in colorectal cancer. CONCLUSIONS: These results reveal the unexpected metabolic rewiring resulting from the CSF-1R receptor targeting of human macrophages and its potential to reverse macrophage-mediated chemoresistance in colorectal tumors.


Asunto(s)
Neoplasias Colorrectales , Macrófagos Asociados a Tumores , Humanos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Macrófagos Asociados a Tumores/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico , Reprogramación Metabólica
19.
ACS Infect Dis ; 10(10): 3553-3576, 2024 Oct 11.
Artículo en Inglés | MEDLINE | ID: mdl-39327729

RESUMEN

The apicoplast is an essential organelle for the viability of apicomplexan parasites Plasmodium falciparum or Toxoplasma gondii, which has been proposed as a suitable drug target for the development of new antiplasmodial drug-candidates. Plasmodione, an antimalarial redox-active lead drug is active at low nM concentrations on several blood stages of Plasmodiumsuch as early rings and gametocytes. Nevertheless, its precise biological targets remain unknown. Here, we described the synthesis and the evaluation of new heteroaromatic analogues of plasmodione, active on asexual blood P. falciparum stages and T. gondii tachyzoites. Using a bioimaging-based analysis, we followed the morphological alterations of T. gondii tachyzoites and revealed a specific loss of the apicoplast upon drug treatment. Lipidomic and fluxomic analyses determined that drug treatment severely impacts apicoplast-hosted FASII activity in T. gondii tachyzoites, further supporting that the apicoplast is a primary target of plasmodione analogues. To follow the drug localization, "clickable" analogues of plasmodione were designed as tools for fluorescence imaging through a Cu(I)-catalyzed azide-alkyne cycloaddition reaction. Short-time incubation of two probes with P. falciparum trophozoites and T. gondii tachyzoites showed that the clicked products localize within, or in the vicinity of, the apicoplast of both Apicomplexa parasites. In P. falciparum, the fluorescence signal was also associated with the mitochondrion, suggesting that bioactivation and activity of plasmodione and related analogues are potentially associated with these two organelles in malaria parasites.


Asunto(s)
Antimaláricos , Apicoplastos , Plasmodium falciparum , Toxoplasma , Apicoplastos/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Toxoplasma/efectos de los fármacos , Antimaláricos/farmacología , Antimaláricos/química , Antimaláricos/síntesis química , Humanos , Imagen Óptica
20.
Int J Parasitol Drugs Drug Resist ; 26: 100565, 2024 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-39332236

RESUMEN

Emergence and spread of parasite resistance to artemisinins, the first-line antimalarial therapy, threaten the malaria eradication policy. To identify therapeutic targets to eliminate artemisinin-resistant parasites, the functioning of the apicoplast and the mitochondrion was studied, focusing on the fatty acid synthesis type II (FASII) pathway in the apicoplast and the electron transfer chain in the mitochondrion. A significant enrichment of the FASII pathway among the up-regulated genes in artemisinin-resistant parasites under dihydroartemisinin treatment was found, in agreement with published transcriptomic data. However, using GC-MS analyzes of fatty acids, we demonstrated for the first time that the FASII pathway is non-functional, ruling out the use of FASII inhibitors to target artemisinin-resistant parasites. Conversely, by assessing the modulation of the oxygen consumption rate, we evidenced that mitochondrial respiration remains functional and flexible in artemisinin-resistant parasites and even at the quiescent stage. Two novel compounds targeting electron transport chain (ELQ300, ELQ400) efficiently killed quiescent artemisinin-resistant parasites. Therefore, mitochondrial respiration represents a key target for the elimination of artemisinin-resistant persistent Plasmodium falciparum parasites.

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