RESUMEN
SUMMARYPlasmodium falciparum, the agent responsible for malaria, is an obligate intracellular protozoan parasite. For proliferation, differentiation and survival, it relies on its own protein-encoding genes, as well as its host cells for nutrient sources. Nutrients and subsequent metabolites are required by the parasites to support their high rate of growth and replication, particularly in the intra-erythrocytic stages of the parasite that are responsible for the clinical symptoms of the disease. Advances in mass spectrometry have improved the analysis of endogenous metabolites and enabled a global approach to identify the parasite's metabolites by the so-called metabolomic analyses. This level of analysis complements the genomic, transcriptomic and proteomic data already available and should allow the identification of novel metabolites, original pathways and networks of regulatory interactions within the parasite, and between the parasite and its hosts. The field of metabolomics is just in its infancy in P. falciparum, hence in this review, we concentrate on the available methodologies and their potential applications for deciphering important biochemical processes of the parasite, such as the astonishingly diverse phospholipid biosynthesis pathways. Elucidating the regulation of the biosynthesis of these crucial metabolites could help design of future anti-malarial drugs.
Asunto(s)
Fosfolípidos/biosíntesis , Plasmodium falciparum/metabolismo , Vías Biosintéticas , Espectrometría de Masas , Metabolómica/métodos , Metabolómica/tendencias , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
A sensitive and selective liquid chromatography-mass spectrometry (LC-MS) method has been developed for the determination of a new antimalarial bisthiazolium salt, SAR97276, in mouse plasma and red blood cells (RBCs). A drug of the same chemical series as the test drug, T2, was used as internal standard. The method involved solid phase extraction of the compound and the internal standard from the two matrices using Oasis HLB columns. LC separation was performed on a Zorbax eclipse XDB C8 column (5 microm) with a mobile phase of acetonitrile containing trimethylamine (130 microl/l, solvent A) and 2 mM ammonium formate buffer (solvent B). MS data were acquired in single ion monitoring mode at m/z 227 for SAR97276 and m/z 326 for T2. The matrix had no influence on the detection of either SAR97276 or T2. The drug/internal standard peak area ratios were linked via quadratic relationships to plasma (1.65-1322 ng/ml) and RBC concentrations (3.31-2644 ng/ml). Precision was below 14% and accuracy was 91.4-104%. Dilution of the samples had no influence on the performance of the method. Extraction recoveries of SAR97276 were > or =90% in plasma and > or =60% in RBCs. The lower limits of quantitation were 1.65 ng/ml in plasma and 3.31 ng/ml in RBCs. Stability tests under various conditions were also investigated. The method was successfully used to determine the pharmacokinetic profile of SAR97276 in healthy mouse.
Asunto(s)
Antimaláricos/sangre , Cromatografía Liquida/métodos , Eritrocitos/química , Espectrometría de Masas/métodos , Tiazoles/sangre , Animales , Antimaláricos/administración & dosificación , Antimaláricos/farmacocinética , Área Bajo la Curva , Cromatografía Liquida/instrumentación , Estabilidad de Medicamentos , Eritrocitos/metabolismo , Femenino , Semivida , Inyecciones Intraperitoneales , Espectrometría de Masas/instrumentación , Tasa de Depuración Metabólica , Ratones , Estructura Molecular , Reproducibilidad de los Resultados , Temperatura , Tiazoles/química , Tiazoles/farmacocinética , Tiazoles/normas , Factores de TiempoRESUMEN
A new class of antimalarial drugs targeting phospholipid metabolism of the malarial parasite is now in development. In the strategy of this development, two mono-thiazolium salts, T1 and T2, need to be monitored. A liquid chromatography-mass spectrometry (LC-MS) method has been developed and validated according to FDA guidelines for simultaneous determination of T1 and T2 in plasma, whole blood and red blood cells (RBCs) from human and rat. The sample-pre-treatment procedure involved solid phase extraction after protein precipitation. Chromatography was carried out on a Zorbax eclipse XDB C8 column and mass spectrometric analysis was performed using an Agilent 1,100 quadrupole mass spectrometer working with an electrospray ionization source. LC-MS data were acquired in single ion monitoring mode at m/z 312, 326 and 227 for T1, T2 and the internal standard (T3), respectively. The drug/internal standard peak area ratios were linked via a quadratic relationship to concentrations (human and rat plasma: 2.25-900 microg/l; human blood and rat RBCs: 4.5-900 microg/kg). Precision was below 14.5% for T1 and below 13% for T2. Accuracy was 92.6-111% for T1 and 95.6-108% for T2. Extraction recoveries were >or=85% in plasma and >or=53% in blood and RBCs. For T1 and T2, the lower limits of quantitation were 2.25 microg/l in plasma, and 4.5 microg/kg in whole blood and RBCs. Stability tests under various conditions were also investigated. This highly specific and sensitive method was useful to analyse samples from pharmacokinetic studies carried out in rat and would also be useful in clinical trials at a later stage.
Asunto(s)
Antimaláricos/sangre , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Tiazoles/sangre , Animales , Antimaláricos/química , Antimaláricos/farmacocinética , Área Bajo la Curva , Calibración , Relación Dosis-Respuesta a Droga , Estabilidad de Medicamentos , Eritrocitos/química , Semivida , Humanos , Tasa de Depuración Metabólica , Estructura Molecular , Plasma/química , Guías de Práctica Clínica como Asunto/normas , Ratas , Ratas Sprague-Dawley , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de Electrospray/métodos , Tiazoles/química , Tiazoles/farmacocinéticaRESUMEN
Plasmodium knowlesi-infected erythrocytes efficiently incorporated choline and metabolize it into phosphatidylcholine via the de novo Kennedy pathway. No formation of either betaine or acetylcholine was detected. At physiological concentrations of external choline, isotopic equilibrium between intracellular choline and phosphocholine was reached in less than 1 h, whereas labeled phosphatidylcholine accumulated constantly, until at least 210 min. During this time, intracellular CDP-choline remained quite low compared to phosphocholine, which suggests that choline-phosphate cytidylyltransferase (EC 2.7.7.15) is the rate-limiting step of the Kennedy pathway. However, this activity was probably not saturated in situ by phosphocholine, since the external choline concentration, up to 100 microM, can regulate phosphatidylcholine biosynthesis via the level of intracellular phosphocholine. This was corroborated by the respective velocities and affinity characteristics of the three enzymatic steps involved in the Kennedy pathway. These results, together with the localization of both choline metabolites and enzyme activities, provide a precise scheme of the dynamics of de novo phosphatidylcholine biosynthesis. Concerning the alternative pathway for phosphatidylcholine biosynthesis via the methylation of phosphatidylethanolamine, we show that an increase in de novo phosphatidylcholine biosynthesis could instigate a concomitant decrease in the steps of phosphatidylethanolamine methylation, indicating that the parasite is able to modulate its phosphatidylcholine biosyntheses.
Asunto(s)
Eritrocitos/parasitología , Malaria/sangre , Nucleotidiltransferasas/sangre , Fosfatidilcolinas/biosíntesis , Animales , Citidililtransferasa de Colina-Fosfato , Haplorrinos , Modelos BiológicosRESUMEN
In its blood stages the malaria parasite, Plasmodium, displays very high lipid metabolism. We present evidence for an abundant long-chain acyl-CoA synthetase (EC 6.2.1.3) activity in Plasmodium knowlesi-infected simian erythrocytes. The activity was found to be 20-fold higher in the schizont-infected (the last parasite stage) than in control erythrocytes. The cosubstrate requirements of the enzyme were similar to those previously reported for acyl-CoA synthetases from other sources. Among the separated reaction products of oleyl-CoA synthetase, only PPi and oleyl-CoA were inhibitory, with Ki over 350 microM. The fatty acid specificity of the parasite acyl-CoA synthetase activity was fairly marked and depended on the unsaturation state of the substrate. The tested fatty acids displayed similar Vmax, whereas their Km ranged from 11 (palmitate) to 59 microM (arachidonate). Finally, experiments involving heat inactivation and separation on hydroxyapatite excluded the presence of a specific arachidonyl-CoA synthetase identical to those present in other cells. On the other hand, fatty acid competition experiments evidenced the existence of at least two distinct enzymatic sites for fatty acid activation in P. knowlesi-infected simian erythrocytes: one is specific for saturated fatty acids and the other for polyunsaturated species, whereas oleate could be activated at both sites.
Asunto(s)
Coenzima A Ligasas/sangre , Eritrocitos/enzimología , Malaria/enzimología , Animales , Eritrocitos/parasitología , Cinética , Macaca fascicularis , Macaca mulatta , Malaria/sangre , Plasmodium/patogenicidad , Valores de Referencia , Especificidad por SustratoRESUMEN
Using capillary gas-liquid chromatography, we have analyzed the alteration in the total fatty acid, phospholipid and neutral lipid compositions of the monkey erythrocyte, after infection by the malarial parasite Plasmodium knowlesi. Data based on fatty acid quantitation show that the phospholipid composition is altered, with particularly large increases in phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the most abundant phospholipids in normal and P. knowlesi-schizont-infected cells. Unesterified fatty acids were found to be less abundant in infected cells. The total fatty acid content of the cell is increased 6-fold during infection, and total fatty acid composition is also changed: the infected cells are richer in palmitate (+23%), oleate (+29%) and linoleate (+89%), but contained less stearate (-27%) and arachidonate (-40%). The determination of the fatty acid composition of individual phospholipids, neutral lipids and unesterified fatty acids showed that choline-containing phospholipids (PC and sphingomyelin) were not as altered in their fatty acid pattern as anionic phospholipids (PE, phosphatidylserine (PS) and phosphatidylinositol (PI) and lysophosphatidylcholine (lysoPC). Specific alterations in the fatty acid compositions of individual phospholipids were detected, whereas the rise in linoleic acid was the only change during infection that was recovered in each phospholipid (except PC), neutral lipid and unesterified fatty acids. The fatty acid composition of the neutral lipids and unesterified fatty acids was particularly modified: the only rise in arachidonic acid level was observed in these lipid classes after infection. The total plasmalogen level of the erythrocyte is decreased in infected cells (-60%), but their level is increased in PI.
Asunto(s)
Eritrocitos/metabolismo , Ácidos Grasos/sangre , Lípidos/sangre , Malaria/sangre , Fosfolípidos/sangre , Animales , Cromatografía de Gases , Eritrocitos/parasitología , Ácidos Grasos no Esterificados/sangre , Glicéridos/sangre , Concentración de Iones de Hidrógeno , Macaca fascicularisRESUMEN
Choline kinase (EC 2.7.1.32) was investigated in plasmodium falciparum-infected erythrocytes. Disrupted infected erythrocytes had a choline kinase activity of 1.9 +/- 0.2 nmol phosphorylcholine/10(7) infected cells per h, whereas the activity in normal uninfected erythrocytes was less than 6 pmol/10(7) cells per h. A broad alkaline optimal pH (7.9-9.2) was observed. The Km values for choline and ATP were 79 +/- 20 microM, and 1.3 +/- 0.3 mM, respectively. ATP concentrations higher than 12 mM inhibited choline kinase. Maximal activity was registered with a Mg2+ concentration of 10 mM, whereas its replacement by Mn2+, or other divalent cations, involved a decrease in choline kinase activity of at least 75%. Inhibition by products of the reaction, such as phosphorylcholine and ADP was investigated. In plasmodium knowlesi-infected erythrocytes, choline kinase had similar properties, but with a much higher specific activity of 16.4 +/- 2.1 nmol/10(7) infected cells per h. Subcellular fractionation of P. knowlesi-infected erythrocyte suspensions revealed that choline kinase was located exclusively in the cytosol of the parasite. We show that this enzyme is a useful index of parasite cytosolic content leakage, when infected erythrocytes are fractionated by saponin lysis or nitrogen decompression.
Asunto(s)
Colina Quinasa/sangre , Eritrocitos/enzimología , Malaria/diagnóstico , Fosfotransferasas/sangre , Plasmodium falciparum/patogenicidad , Pruebas Enzimáticas Clínicas , Humanos , Concentración de Iones de Hidrógeno , Cinética , Malaria/sangre , Malaria/enzimologíaRESUMEN
CDPcholine: 1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and CDPethanolamine: 1,2-diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) activities were investigated in Plasmodium knowlesi-infected erythrocytes obtained from Macaca fascicularis monkeys. Disrupted infected erythrocytes possess a cholinephosphotransferase activity (1.3 +/- 0.2 nmol phosphatidylcholine/10(7) infected cells per h) 1.5-times higher than the ethanolaminephosphotransferase activity. Optimal activities of both enzymes were observed in the presence of 12 mM MnCl2, which was about 3-times as effective as 40 mM MgCl2 as a cofactor. The two activities had similar dependences on pH and thermal inactivation. Their Arrhenius plots show an identical break at 17 degrees C and the corresponding activation energies below and above the critical temperature were similar for the two activities. Sodium deoxycholate, sodium dodecyl sulfate, Triton X-100, beta-D-octylglucoside and lysophosphatidylcholine strongly inhibited the two activities above their critical micellar concentration, but the first three detergents stimulated the activities at lower concentrations. Saponin (0.004-0.5%) either did not affect the two activities or else increased them. Cholinephosphotransferase and ethanolaminephosphotransferase activities had apparent Km values for the CDP ester of 23.4 and 18.6 microM, respectively. CDPcholine and CDPethanolamine competitively inhibited the ethanolaminephosphotransferase and cholinephosphotransferase activities, respectively. The high selectivity of these activities for individual molecular species of diradylglycerol suggests that substrate specificity is responsible for the various molecular species of Plasmodium-infected erythrocyte phospholipids. However, cholinephosphotransferase and ethanolaminephosphotransferase had different dependences on 1,2-dilauroylglycerol and 1-oleylglycerol, which were substrates for cholinephosphotransferase but not for ethanolaminephosphotransferase under our conditions. These data provide the first characterization of an enzyme involved in the intense lipid metabolism in Plasmodium-infected erythrocytes, and the presence of cholinephosphotransferase demonstrates a biosynthesis of phosphatidylcholine by the Kennedy pathway after infection. Our data suggest that cholinephosphotransferase and ethanolaminephosphotransferase activities could be catalyzed by the same enzyme. Furthermore, since host erythrocytes are devoid of these enzymatic activities, cholinephosphotransferase is a parasite-specific membrane-associated enzyme which can be used as a probe or marker.
Asunto(s)
Diacilglicerol Colinafosfotransferasa/sangre , Eritrocitos/enzimología , Etanolaminofosfotransferasa/sangre , Fosfotransferasas/sangre , Plasmodium/enzimología , Animales , Pruebas Enzimáticas Clínicas , Cinética , Macaca fascicularis , Macaca mulatta , Malaria/diagnóstico , Malaria/enzimología , Plasmodium/patogenicidadRESUMEN
Using high performance liquid chromatography and gas-liquid chromatography, we have characterized the phosphatidylcholine and phosphatidylethanolamine molecular species composition of trophozoite and schizont forms of Plasmodium knowlesi parasitized erythrocytes. Similarly, we determined these parameters in the erythrocyte membranes of trophozoite parasitized cells, unparasitized erythrocytes from infected monkeys before and after a chloroquine treatment and erythrocytes from monkeys that had never been infected. Plasma phosphatidylcholine molecular species composition was also studied. P. knowlesi parasitized erythrocytes presented higher amounts of 16:0/18:2-phosphatidylcholine than the various control cells, which appeared to be compensated for by a decrease in 18:0/20:4-, 16:0/20:3-, 16:0/18:1-, 18:0/18:2-, 18:0/20:3-, 16:0/16:0- and 16:0/18:0-phosphatidylcholines. In the case of phosphatidylethanolamine, the alterations were quantitatively of greater importance and consisted of an increase in, again, 16:0/18:2-phosphatidylethanolamine and a decrease in several species containing 20:4, namely 16:0/20:4-, 18:0/20:4- and 18:1/20:4-phosphatidylethanolamine; also the levels of alkoxy-phosphatidylethanolamines were markedly decreased. P. knowlesi development within monkey erythrocytes therefore appears to be associated with changes in phosphatidylcholine and phosphatidylethanolamine molecular species in the whole parasitized cell. These alterations are also exhibited by the host cell membrane, which provides the first experimental evidence that the parasite is able to manipulate the erythrocyte membrane lipid species composition. The consequences of these alterations on membrane physiology are discussed, as well as the implications that these data may have on the trafficking of phosphatidylcholine and phosphatidylethanolamine in the erythrocytes of P. knowlesi infected monkeys.
Asunto(s)
Eritrocitos/metabolismo , Malaria/sangre , Fosfatidilcolinas/sangre , Fosfatidiletanolaminas/sangre , Plasmodium/fisiología , Animales , Cloroquina/uso terapéutico , Membrana Eritrocítica/metabolismo , Eritrocitos/parasitología , Ácidos Grasos/sangre , Macaca fascicularis , Macaca mulatta , Malaria/tratamiento farmacológicoRESUMEN
Pig pancreatic phospholipase A2 does not act on normal erythrocytes, but the membrane penetrating capacity is enhanced by the covalent attachment of one fatty acyl chain to Lys-116 of the enzyme. Taking advantage of the impaired packing of phospholipids in the membrane of Plasmodium infected erythrocytes it was demonstrated that a lauric acid derivative of phospholipase A2 is capable of exclusively attaching the infected erythrocytes in vitro, leaving the uninfected cells undisturbed. The chemically modified phospholipase A2 appeared to cause death of the parasite in cell cultures of infected erythrocytes.
Asunto(s)
Eritrocitos/parasitología , Malaria/sangre , Fosfolipasas A/metabolismo , Fosfolipasas/metabolismo , Animales , Hemólisis , Técnicas In Vitro , Macaca fascicularis , Macaca mulatta , Fosfatidilcolinas/farmacología , Fosfolipasas A2RESUMEN
In Plasmodium falciparum-infected erythrocyte homogenates, the specific activity of ethanolamine kinase (7.6 +/- 1.4 nmol phosphoethanolamine/10(7) infected cells per h) was higher than choline kinase specific activity (1.9 +/- 0.2 nmol phosphocholine/10(7) infected cells per h). The Km of choline kinase for choline was 79 +/- 20 microM, and ethanolamine was a weak competitive inhibitor of the reaction (Ki = 92 mM). Ethanolamine kinase had a Km for ethanolamine of 188 +/- 19 microM, and choline was a competitive inhibitor of ethanolamine kinase with a very high Ki of 268 mM. Hemicholinium 3 inhibited choline kinase activity, but had no effect on ethanolamine kinase activity. In contrast, D-2-amino-1-butanol selectively inhibited ethanolamine kinase activity. Furthermore, when the two enzymes were subjected to heat inactivation, 85% of the choline kinase activity was destroyed after 5 min at 50 degrees C, whereas ethanolamine kinase activity was not altered. Our results indicate that the phosphorylation of choline and ethanolamine was catalyzed by two distinct enzymes. The presence of a de novo phosphatidylethanolamine Kennedy pathway in P. falciparum contributes to the bewildering variety of phospholipid biosynthetic pathways in this parasitic organism.
Asunto(s)
Colina Quinasa/análisis , Colina/metabolismo , Etanolaminas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol) , Fosfotransferasas/análisis , Plasmodium falciparum/enzimología , Amino Alcoholes/farmacología , Unión Competitiva , Etanolamina , Calor , Cinética , Fosforilación , Factores de TiempoRESUMEN
The uptake of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) in Plasmodium knowlesi infected erythrocytes has been studied. Whereas uptake of phospholipids, in the absence of phospholipid transfer proteins, is negligible in control cells, the infected cells can incorporate considerable amounts of added phospholipids. The uptake is enhanced by the presence of lipid transfer proteins. Doubly labeled [3H]oleate, [14C]choline) PC does not undergo any appreciable remodelling following uptake, which strongly suggests that plasma PC is used as such for the biogenesis of the parasite membranes. Transport of extracellularly offered PS and PE towards the intraerythrocytic parasite and utilization of these lipids by the parasite are confirmed by the observation that these lipids are converted into respectively PE and PC. The extent and rate of these conversions depend on the way the phospholipids are introduced into the infected cells.
Asunto(s)
Proteína de Unión a Andrógenos , Membrana Eritrocítica/metabolismo , Malaria/sangre , Fosfolípidos/sangre , Animales , Proteínas Portadoras/sangre , Eritrocitos/parasitología , Macaca fascicularis , Macaca mulatta , Fosfatidilcolinas/sangre , Fosfatidiletanolaminas/sangre , Fosfatidilserinas/sangre , Proteínas de Transferencia de Fosfolípidos , Plasmodium/crecimiento & desarrollo , Plasmodium/fisiologíaRESUMEN
Large-scale in vitro screening of different types of ionophores previously pinpointed nine compounds that were very active and selective in vitro against Plasmodium falciparum; their in vitro and in vivo antimalarial effects were further studied. Addition of the ionophores to synchronized P. falciparum suspensions revealed that all P. falciparum stages were sensitive to the drugs. However, the schizont stages were three- to ninefold more sensitive, and 12 h was required for complete parasite clearance. Pretreatment of healthy erythrocytes with toxic doses of ionophores for 24 to 48 h showed that the activity was not due to an irreversible effect on the host erythrocyte. No preferential ionophore adsorption in infected or uninfected erythrocytes occurred. On the other hand, ionophore molecules strongly bound to serum proteins since increasing the serum concentration from 2 to 50% led to almost a 25-fold parallel increase in the ionophore 50% inhibitory concentration. Mice infected with the malaria parasites Plasmodium vinckei petteri or Plasmodium chabaudi were successfully treated with eight ionophores in a 4-day suppressive test. The 50% effective dose after intraperitoneal administration ranged from 0.4 to 4.1 mg/kg of body weight, and the therapeutic indices were about 5 for all ionophores except monensin A methyl ether, 5-bromo lasalocid A, and gramicidin D, whose therapeutic indices were 12, 18, and 344, respectively. These three compounds were found to be curative, with no recrudescence. Gramicidin D, which presented impressive antimalarial activity, requires parenteral administration, while 5-bromo lasalocid A has the major advantage of being active after oral administration. Overall, the acceptable levels of toxicity and the good in vivo therapeutic indices in the rodent model highlight the interesting potential of these ionophores for the treatment of malaria in higher animals.
Asunto(s)
Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Ionóforos/farmacología , Ionóforos/uso terapéutico , Malaria/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Animales , Antimaláricos/sangre , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Hematócrito , Humanos , Ionóforos/sangre , Malaria/sangre , Malaria/parasitología , Masculino , Ratones , Plasmodium falciparum/crecimiento & desarrolloRESUMEN
The intraerythrocytic malaria parasite depends on the surrounding medium for a supply of phospholipid precursors. Efficient inhibition (IC50 7-90 microM) of Plasmodium falciparum growth in vitro was achieved using modified fatty acids. The fatty acid analogues most effective in suppressing P. falciparum growth in culture were also the most active inhibitors of acyl-CoA synthetase from the monkey parasite P. knowlesi.
Asunto(s)
Coenzima A Ligasas/antagonistas & inhibidores , Ácidos Grasos/farmacología , Ácidos Palmíticos/farmacología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium/enzimología , Animales , Células Cultivadas , Óxidos N-Cíclicos/farmacología , Eritrocitos/parasitología , Humanos , Palmitatos/farmacología , Plasmodium falciparum/efectos de los fármacos , Ácidos Ricinoleicos/farmacologíaRESUMEN
Plasmodium knowlesi-infected erythrocytes possess a membranous cytidine 5'-diphospho-1,2-diacyl-sn-glycerol: myoinositol 3-phosphatidyl transferase (PI synthase) (EC 2.7.8.11) activity of 10 +/- 1.7 nmol min-1 per 10(10) infected cells. The activity was successfully solubilized with 40 mM n-octyl-beta-D-glucopyranoside in the presence of bivalent metal ions which were absolutely required for activity. The optimal pH was 8 and the apparent Ks for Mn2+ was 0.1 mM. Mg2+ allowed two-fold higher PI synthase activity, with an optimum above 100 mM. Calcium alone was ineffective while at 2 mM it inhibited solubilized PI synthase activity in the presence of 100 mM Mg2+. Enzymatic activity was fully dependent on CDP-diacylglycerol and inositol with apparent Km of 0.16 +/- 0.1 mM and 1 +/- 0.5 mM respectively. Affinity chromatography clearly showed CDP-diacylglycerol-dependent interactions of PI synthase with CDP-diacylglycerol Sepharose. However, elution of enzymatic activity in an active form was unsuccessful while SDS-PAGE of the eluate showed one apparent band. Incubations of Plasmodium falciparum-infected erythrocytes with 32P or [3H]inositol revealed de novo biosynthesis of phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate which appeared to predominate in the second half of the asexual cellular cycle. Ionomycin, a calcium ionophore, induced Li(+)-sensitive production of radioactive inositol phosphates, with neo-synthesized inositol 1,4,5-trisphosphate accumulation being the highest.
Asunto(s)
Eritrocitos/enzimología , Eritrocitos/parasitología , Fosfatos de Fosfatidilinositol/metabolismo , Plasmodium/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/aislamiento & purificación , Animales , CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferasa , Calcio/farmacología , Cationes Bivalentes/farmacología , Citidina Difosfato Diglicéridos/metabolismo , Macaca fascicularis , Plasmodium/enzimología , Plasmodium/crecimiento & desarrollo , Plasmodium falciparum/enzimología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Plasmodium knowlesi/enzimología , Plasmodium knowlesi/crecimiento & desarrollo , Plasmodium knowlesi/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/efectos de los fármacosRESUMEN
D vitamins are effective inhibitors of the in vitro intraerythrocytic growth of Plasmodium falciparum. Disappearance of the parasitemia was observed after 48 h contact between infected cells and 5 x 10(-6) M 1 alpha-hydroxycholecalciferol, 5 x 10(-5) M 25-hydroxycholecalciferol (25-OH-D-3), 1 alpha, 25-dihydroxycholecalciferol or 2.5 x 10(-4) vitamin D-2 and D-3. A 48 h pretreatment of healthy erythrocytes with 5 x 10(-5) M 25-OH-D-3 did not change their susceptibility to invasion by the parasite and their ability to support the growth of P. falciparum. Ionomycin, a calcium ionophore, and EGTA prevented parasite development at concentrations greater than 2 x 10(-7) M and 4 x 10(-4) M, respectively, but did not antagonize the inhibitory activity of 25-OH-D-3. Addition of 25-OH-D-3 for 12 or 24 h duration to synchronized cultures, showed that the drug had a schizonticidal action, but was without effect when parasites were in the ring form.
Asunto(s)
Colecalciferol/farmacología , Ergocalciferoles/farmacología , Eritrocitos/parasitología , Hidroxicolecalciferoles/farmacología , Plasmodium falciparum/crecimiento & desarrollo , Animales , Calcifediol , Calcitriol/farmacología , Relación Dosis-Respuesta a Droga , Ácido Egtácico/farmacología , Eritrocitos/efectos de los fármacos , Éteres/farmacología , Humanos , Ionomicina , Plasmodium falciparum/efectos de los fármacosRESUMEN
Metabolic pathways leading to lipid biosynthesis in four different developmental stages of Schistosoma mansoni were explored and quantified by incubation in the presence of labeled precursors in a chemically defined medium. At the schistosomulum stage and in male, female, or paired worms, glycerol and oleate incorporation into neutral lipids, mainly in the form of triacylglycerols, was greater than into phospholipids, whereas in 11-and 15-day-old worms, synthesis mainly led to phospholipids. Incorporation into phospholipids was recovered largely in phosphatidylcholine, and distribution into other phospholipids depended on the developmental stage. Incorporation of choline and ethanolamine into their respective phospholipids represented up to 15% of the parasitic phospholipid content. The formation of phosphatidylcholine by phosphatidylethanolamine methylation occurred mainly in the immature parasitic stages. Inositol incorporation was also measurable, whereas [14C]serine incorporation was low or undetectable. Addition of 1-palmitoyl-2-[14C]oleyl phosphatidylcholine revealed a very high uptake of this phospholipid by the immature stages but further metabolism was not detectable. In contrast, adult S. mansoni were completely unable to take up or absorb this exogenous phospholipid. The most striking aspect of this study was the relatively high metabolic activity in 11-day-old worms and the lower but sustained activity on day 15 and at the schistosomulum stage. By comparison, biosynthetic activity in adult S. mansoni, on which research studies have been focused until now, was very low. We also discuss the participation of lipid metabolism in the constant renewal of the membrane complex which is essential to parasitism by S. mansoni.
Asunto(s)
Lípidos de la Membrana/metabolismo , Schistosoma mansoni/metabolismo , Envejecimiento , Animales , Biomphalaria , Radioisótopos de Carbono , Colina/metabolismo , Etanolamina , Etanolaminas/metabolismo , Femenino , Glicerol/metabolismo , Inositol/metabolismo , Masculino , Fosfolípidos/metabolismo , Surfactantes Pulmonares/metabolismo , Schistosoma mansoni/crecimiento & desarrollo , Serina/metabolismo , Factores Sexuales , TritioRESUMEN
The cholesterol synthesis of rhesus monkey erythrocytes parasitized by Plasmodium knowlesi and human erythrocytes infected by P. falciparum, as measured by incorporation of [1-14C]acetate and 3H2O, was almost undetectable, concordant with very low levels of measurable 3-hydroxy-3-methyl glutaryl-CoA reductase activity. In addition, both types of infected cells exchanged cholesterol with the plasma at the same rate as uninfected cells. The data do not exclude the possibility of cholesterol transfer from uninfected to infected cells.
Asunto(s)
Colesterol/sangre , Eritrocitos/metabolismo , Plasmodium/metabolismo , Animales , Transporte Biológico , Colesterol/biosíntesis , Eritrocitos/parasitología , Interacciones Huésped-Parásitos , Humanos , Macaca mulatta/metabolismo , Lípidos de la Membrana/metabolismo , Plasmodium falciparum/metabolismo , Especificidad de la EspecieRESUMEN
A number of choline and ethanolamine analogs were evaluated as inhibitors of P. falciparum growth in vitro. 1-Aziridineethanol, DL-2-amino-1,3-propranediol and D- or L-2-amino-1-butanol were the most efficient inhibitors of parasite multiplication, with an IC50 of 50-80 microM, whereas numerous other analogs were less active. The effect of D-2-amino-1-butanol on various metabolisms of P. knowlesi-infected simian erythrocytes was studied by incubating these cells with different labeled precursors of phospholipids, nucleic acids, proteins, and with radioactive glucose. In the presence of radioactive glycerol, oleate or lysophosphatidylcholine, the appearance of radioactivity in an unnatural phospholipid indicated that 2-aminobutanol was incorporated into a new PL which accounted for up to 30-40% of the total biosynthesized lipids. This new phospholipid accumulated primarily at the expense of PE biosynthesis and decreased the decarboxylation of phosphatidylserine. These effects were not accompanied, over a large range of concentrations, by any parallel change in nucleic or protein synthesis, nor in glucose metabolism. These data demonstrate that the incorporation of analogs, instead of the natural polar head groups, into cellular phospholipids, and/or modification of phospholipid composition have a deleterious impact on the growth of Plasmodium. It follows that PL metabolism is a crucial process for Plasmodium growth and may constitute a potentially fruitful chemotherapeutic approach to malaria.
Asunto(s)
Amino Alcoholes/farmacología , Antimaláricos/farmacología , Fosfolípidos/metabolismo , Animales , Colina/metabolismo , Relación Dosis-Respuesta a Droga , Glicerol/metabolismo , Lípidos de la Membrana/metabolismo , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/metabolismo , Serina/metabolismo , Relación Estructura-ActividadRESUMEN
The effect of the antimalarial drug chloroquine (CQ) on the phospholipid metabolism in Plasmodium knowlesi-infected simian erythrocytes has been studied by incubating cells with different labeled precursors and various concentrations of CQ. The drug induced considerable modifications of this metabolism but at the same time decreased nucleic acid and protein synthesis as well as the output of 14CO2 from radioactive glucose. Phosphatidylcholine biosynthesis was severely reduced. However, under these conditions, CQ had the early effect of markedly increasing phosphatidylinositol labeling from radioactive inositol, fatty acids, 1-(14C)palmitoyl-lysophosphatidylcholine, but not from glycerol. Synthesis of phosphatidylserine from (14C)serine and of phosphatidylethanolamine from labeled glycerol, ethanolamine, and serine was increased, especially at high CQ concentrations when the whole metabolism of the parasite was severely reduced. These effects reflect a deep differential effect of CQ on the intense phospholipid metabolism of the Plasmodium-infected erythrocytes, which might involve a redirecting of phospholipid metabolism similar to that induced by other cationic amphiphilic drugs, and a compensatory synthesis resulting from the severe blockage of phosphatidylcholine synthesis.