RESUMEN
In the developing seeds of all higher plants, filial cells are symplastically isolated from the maternal tissue supplying photosynthate to the reproductive structure. Photoassimilates must be transported apoplastically, crossing several membrane barriers, a process facilitated by sugar transporters. Sugars Will Eventually be Exported Transporters (SWEETs) have been proposed to play a crucial role in apoplastic sugar transport during phloem unloading and the post-phloem pathway in sink tissues. Evidence for this is presented here for developing seeds of the C4 model grass Setaria viridis. Using immunolocalization, SvSWEET4 was detected in various maternal and filial tissues within the seed along the sugar transport pathway, in the vascular parenchyma of the pedicel, and in the xylem parenchyma of the stem. Expression of SvSWEET4a in Xenopus laevis oocytes indicated that it functions as a high-capacity glucose and sucrose transporter. Carbohydrate and transcriptional profiling of Setaria seed heads showed that there were some developmental shifts in hexose and sucrose content and consistent expression of SvSWEET4 homologues. Collectively, these results provide evidence for the involvement of SWEETs in the apoplastic transport pathway of sink tissues and allow a pathway for post-phloem sugar transport into the seed to be proposed.
Asunto(s)
Setaria (Planta) , Azúcares , Azúcares/metabolismo , Setaria (Planta)/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Floema/metabolismo , Transporte Biológico , Proteínas de Transporte de Membrana/metabolismo , Sacarosa/metabolismoRESUMEN
Polymorphisms in the Plasmodium falciparum multidrug resistance protein 1 (pfmdr1) gene and the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene alter the malaria parasite's susceptibility to most of the current antimalarial drugs. However, the precise mechanisms by which PfMDR1 contributes to multidrug resistance have not yet been fully elucidated, nor is it understood why polymorphisms in pfmdr1 and pfcrt that cause chloroquine resistance simultaneously increase the parasite's susceptibility to lumefantrine and mefloquine-a phenomenon known as collateral drug sensitivity. Here, we present a robust expression system for PfMDR1 in Xenopus oocytes that enables direct and high-resolution biochemical characterizations of the protein. We show that wild-type PfMDR1 transports diverse pharmacons, including lumefantrine, mefloquine, dihydroartemisinin, piperaquine, amodiaquine, methylene blue, and chloroquine (but not the antiviral drug amantadine). Field-derived mutant isoforms of PfMDR1 differ from the wild-type protein, and each other, in their capacities to transport these drugs, indicating that PfMDR1-induced changes in the distribution of drugs between the parasite's digestive vacuole (DV) and the cytosol are a key driver of both antimalarial resistance and the variability between multidrug resistance phenotypes. Of note, the PfMDR1 isoforms prevalent in chloroquine-resistant isolates exhibit reduced capacities for chloroquine, lumefantrine, and mefloquine transport. We observe the opposite relationship between chloroquine resistance-conferring mutations in PfCRT and drug transport activity. Using our established assays for characterizing PfCRT in the Xenopus oocyte system and in live parasite assays, we demonstrate that these PfCRT isoforms transport all 3 drugs, whereas wild-type PfCRT does not. We present a mechanistic model for collateral drug sensitivity in which mutant isoforms of PfMDR1 and PfCRT cause chloroquine, lumefantrine, and mefloquine to remain in the cytosol instead of sequestering within the DV. This change in drug distribution increases the access of lumefantrine and mefloquine to their primary targets (thought to be located outside of the DV), while simultaneously decreasing chloroquine's access to its target within the DV. The mechanistic insights presented here provide a basis for developing approaches that extend the useful life span of antimalarials by exploiting the opposing selection forces they exert upon PfCRT and PfMDR1.
Asunto(s)
Antimaláricos , Malaria Falciparum , Parásitos , Animales , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Cloroquina/metabolismo , Cloroquina/farmacología , Cloroquina/uso terapéutico , Resistencia a Medicamentos/genética , Resistencia a Múltiples Medicamentos , Lumefantrina/farmacología , Lumefantrina/uso terapéutico , Malaria Falciparum/parasitología , Mefloquina/metabolismo , Mefloquina/farmacología , Mefloquina/uso terapéutico , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/uso terapéutico , Parásitos/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Photosynthetic efficiency and sink demand are tightly correlated with rates of phloem loading, where maintaining low cytosolic sugar concentrations is paramount to prevent the downregulation of photosynthesis. Sugars Will Eventually be Exported Transporters (SWEETs) are thought to have a pivotal role in the apoplastic phloem loading of C4 grasses. SWEETs have not been well studied in C4 species, and their investigation is complicated by photosynthesis taking place across two cell types and, therefore, photoassimilate export can occur from either one. SWEET13 homologues in C4 grasses have been proposed to facilitate apoplastic phloem loading. Here, we provide evidence for this hypothesis using the C4 grass Setaria viridis. Expression analyses on the leaf gradient of C4 species Setaria and Sorghum bicolor show abundant transcript levels for SWEET13 homologues. Carbohydrate profiling along the Setaria leaf shows total sugar content to be significantly higher in the mature leaf tip compared with the younger tissue at the base. We present the first known immunolocalization results for SvSWEET13a and SvSWEET13b using novel isoform-specific antisera. These results show localization to the bundle sheath and phloem parenchyma cells of both minor and major veins. We further present the first transport kinetics study of C4 monocot SWEETs by using a Xenopus laevis oocyte heterologous expression system. We demonstrate that SvSWEET13a and SvSWEET13b are high-capacity transporters of glucose and sucrose, with a higher apparent Vmax for sucrose, compared with glucose, typical of clade III SWEETs. Collectively, these results provide evidence for an apoplastic phloem loading pathway in Setaria and possibly other C4 species.
Asunto(s)
Transporte Biológico/genética , Glucosa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Floema/metabolismo , Setaria (Planta)/genética , Setaria (Planta)/metabolismo , Sacarosa/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Fotosíntesis , Transcripción GenéticaRESUMEN
Toxoplasma gondii and Plasmodium falciparum parasites both extrude L-lactate, a byproduct of glycolysis. The P. falciparum Formate Nitrite Transporter, PfFNT, mediates L-lactate transport across the plasma membrane of P. falciparum parasites and has been validated as a drug target. The T. gondii genome encodes three FNTs that have been shown to transport L-lactate, and which are proposed to be the targets of several inhibitors of T. gondii proliferation. Here, we show that each of the TgFNTs localize to the T. gondii plasma membrane and are capable of transporting L-lactate across it, with TgFNT1 making the primary contribution to L-lactate transport during the disease-causing lytic cycle of the parasite. We use the Xenopus oocyte expression system to provide direct measurements of L-lactate transport via TgFNT1. We undertake a genetic analysis of the importance of the tgfnt genes for parasite proliferation, and demonstrate that all three tgfnt genes can be disrupted individually and together without affecting the lytic cycle under in vitro culture conditions. Together, our experiments identify the major lactate transporter in the disease causing stage of T. gondii, and reveal that this transporter is not required for parasite proliferation, indicating that TgFNTs are unlikely to be targets for anti-Toxoplasma drugs.
Asunto(s)
Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Animales , Membrana Celular/metabolismo , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Oocitos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Protozoarias/genética , Toxoplasma/crecimiento & desarrollo , Xenopus/crecimiento & desarrolloRESUMEN
Due to the surge in resistance to common therapies, malaria remains a significant concern to human health worldwide. In chloroquine (CQ)-resistant (CQ-R) strains of Plasmodium falciparum, CQ and related drugs are effluxed from the parasite's digestive vacuole (DV). This process is mediated by mutant isoforms of a protein called CQ resistance transporter (PfCRT). CQ-R strains can be partially re-sensitized to CQ by verapamil (VP), primaquine (PQ) and other compounds, and this has been shown to be due to the ability of these molecules to inhibit drug transport via PfCRT. We have previously developed a series of clotrimazole (CLT)-based antimalarial agents that possess inhibitory activity against PfCRT (4a,b). In our endeavor to develop novel PfCRT inhibitors, and to perform a structure-activity relationship analysis, we synthesized a new library of analogues. When the benzhydryl system was linked to a 4-aminoquinoline group (5a-f) the resulting compounds exhibited good cytotoxicity against both CQ-R and CQ-S strains of P. falciparum. The most potent inhibitory activity against the PfCRT-mediated transport of CQ was obtained with compound 5k. When compared to the reference compound, benzhydryl analogues of PQ (5i,j) showed a similar activity against blood-stage parasites, and a stronger in vitro potency against liver-stage parasites. Unfortunately, in the in vivo transmission blocking assays, 5i,j were inactive against gametocytes.
Asunto(s)
Antimaláricos/farmacología , Compuestos de Bencidrilo/farmacología , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Anopheles , Antimaláricos/síntesis química , Compuestos de Bencidrilo/síntesis química , Cloroquina/farmacología , Diseño de Fármacos , Farmacorresistencia Microbiana/efectos de los fármacos , Femenino , Células Hep G2 , Humanos , Proteínas de Transporte de Membrana , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Células 3T3 NIH , Pruebas de Sensibilidad Parasitaria , Isoformas de Proteínas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , XenopusRESUMEN
Understanding evolution requires detailed knowledge of genotype-phenotype maps; however, it can be a herculean task to measure every phenotype in a combinatorial map. We have developed a computational strategy to predict the missing phenotypes from an incomplete, combinatorial genotype-phenotype map. As a test case, we used an incomplete genotype-phenotype dataset previously generated for the malaria parasite's 'chloroquine resistance transporter' (PfCRT). Wild-type PfCRT (PfCRT3D7) lacks significant chloroquine (CQ) transport activity, but the introduction of the eight mutations present in the 'Dd2' isoform of PfCRT (PfCRTDd2) enables the protein to transport CQ away from its site of antimalarial action. This gain of a transport function imparts CQ resistance to the parasite. A combinatorial map between PfCRT3D7 and PfCRTDd2 consists of 256 genotypes, of which only 52 have had their CQ transport activities measured through expression in the Xenopus laevis oocyte. We trained a statistical model with these 52 measurements to infer the CQ transport activity for the remaining 204 combinatorial genotypes between PfCRT3D7 and PfCRTDd2. Our best-performing model incorporated a binary classifier, a nonlinear scale, and additive effects for each mutation. The addition of specific pairwise- and high-order-epistatic coefficients decreased the predictive power of the model. We evaluated our predictions by experimentally measuring the CQ transport activities of 24 additional PfCRT genotypes. The R2 value between our predicted and newly-measured phenotypes was 0.90. We then used the model to probe the accessibility of evolutionary trajectories through the map. Approximately 1% of the possible trajectories between PfCRT3D7 and PfCRTDd2 are accessible; however, none of the trajectories entailed eight successive increases in CQ transport activity. These results demonstrate that phenotypes can be inferred with known uncertainty from a partial genotype-phenotype dataset. We also validated our approach against a collection of previously published genotype-phenotype maps. The model therefore appears general and should be applicable to a large number of genotype-phenotype maps.
Asunto(s)
Genotipo , Fenotipo , Animales , Modelos Biológicos , Mutación , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , IncertidumbreRESUMEN
The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is a key contributor to multidrug resistance and is also essential for the survival of the malaria parasite, yet its natural function remains unresolved. We identify host-derived peptides of 4-11 residues, varying in both charge and composition, as the substrates of PfCRT in vitro and in situ, and show that PfCRT does not mediate the non-specific transport of other metabolites and/or ions. We find that drug-resistance-conferring mutations reduce both the peptide transport capacity and substrate range of PfCRT, explaining the impaired fitness of drug-resistant parasites. Our results indicate that PfCRT transports peptides from the lumen of the parasite's digestive vacuole to the cytosol, thereby providing a source of amino acids for parasite metabolism and preventing osmotic stress of this organelle. The resolution of PfCRT's native substrates will aid the development of drugs that target PfCRT and/or restore the efficacy of existing antimalarials.
Asunto(s)
Antimaláricos/farmacología , Cloroquina/farmacología , Proteínas de Transporte de Membrana/metabolismo , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Transporte Biológico Activo , Resistencia a Medicamentos/genética , Femenino , Interacciones Huésped-Parásitos/genética , Interacciones Huésped-Parásitos/fisiología , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/metabolismo , Malaria Falciparum/parasitología , Proteínas de Transporte de Membrana/genética , Modelos Biológicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oligopéptidos/metabolismo , Oocitos/metabolismo , Plasmodium falciparum/genética , Transporte de Proteínas , Proteínas Protozoarias/genética , Xenopus laevisRESUMEN
OBJECTIVES: This paper reviews the precision public health literature pertaining to oral health, identifies possible threats that could inadvertently increase health inequities, and proposes potential opportunities that precision public health could utilize to reduce oral health inequities. METHODS: The health sciences literature was reviewed and supplemented with new data to identify important issues relating to precision medicine, precision oral health, precision public health, and health equity. RESULTS: Examples from general health and oral health were provided to illustrate salient concepts. CONCLUSIONS: Future precision public health should utilize multifactorial, multi-level conceptual frameworks and conceptual causal models with upstream social determinants and downstream health effects, as well as a proportionate universalism perspective; and proper analytic methods, including sufficient sample sizes, appropriate statistical competitors, health disparity indices, causal modeling, and internal and external validation.
Asunto(s)
Equidad en Salud , Salud Pública , Disparidades en el Estado de Salud , Modelos Teóricos , Salud BucalRESUMEN
The deployment of artemisinin-based combination therapies (ACTs) has been, and continues to be, integral to reducing the number of malaria cases and deaths. However, their efficacy is being increasingly jeopardized by the emergence and spread of parasites that are resistant (or partially resistant) to the artemisinin derivatives and to their partner drugs, with the efficacy of the latter being especially crucial for treatment success. A detailed understanding of the genetic determinants of resistance to the ACT partner drugs, and the mechanisms by which they mediate resistance, is required for the surveillance of molecular markers and to optimize the efficacy and lifespan of the partner drugs through resistance management strategies. We summarize new insights into the molecular basis of parasite resistance to the ACTs, such as recently-uncovered determinants of parasite susceptibility to the artemisinin derivatives, piperaquine, lumefantrine, and mefloquine, and outline the mechanisms through which polymorphisms in these determinants may be conferring resistance.
Asunto(s)
Artemisininas/farmacología , Artemisininas/uso terapéutico , Resistencia a Medicamentos/efectos de los fármacos , Malaria/tratamiento farmacológico , Parásitos/efectos de los fármacos , Animales , HumanosRESUMEN
In this study the 'Malaria Box' chemical library comprising 400 compounds with antiplasmodial activity was screened for compounds that perturb the internal pH of the malaria parasite, Plasmodium falciparum. Fifteen compounds induced an acidification of the parasite cytosol. Two of these did so by inhibiting the parasite's formate nitrite transporter (PfFNT), which mediates the H+-coupled efflux from the parasite of lactate generated by glycolysis. Both compounds were shown to inhibit lactate transport across the parasite plasma membrane, and the transport of lactate by PfFNT expressed in Xenopus laevis oocytes. PfFNT inhibition caused accumulation of lactate in parasitised erythrocytes, and swelling of both the parasite and parasitised erythrocyte. Long-term exposure of parasites to one of the inhibitors gave rise to resistant parasites with a mutant form of PfFNT that showed reduced inhibitor sensitivity. This study provides the first evidence that PfFNT is a druggable antimalarial target.
Asunto(s)
Antimaláricos/farmacología , Eritrocitos/parasitología , Malaria Falciparum/metabolismo , Transportadores de Ácidos Monocarboxílicos/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Animales , Antimaláricos/química , Transporte Biológico/efectos de los fármacos , Cromatografía Liquida , Evaluación Preclínica de Medicamentos , Humanos , Malaria Falciparum/parasitología , Espectrometría de Masas , Plasmodium falciparum/metabolismo , Plasmodium falciparum/parasitología , Proteínas Protozoarias/metabolismo , Xenopus laevisRESUMEN
Mutations in the Plasmodium falciparum 'chloroquine resistance transporter' (PfCRT) confer resistance to chloroquine (CQ) and related antimalarials by enabling the protein to transport these drugs away from their targets within the parasite's digestive vacuole (DV). However, CQ resistance-conferring isoforms of PfCRT (PfCRTCQR) also render the parasite hypersensitive to a subset of structurally-diverse pharmacons. Moreover, mutations in PfCRTCQR that suppress the parasite's hypersensitivity to these molecules simultaneously reinstate its sensitivity to CQ and related drugs. We sought to understand these phenomena by characterizing the functions of PfCRTCQR isoforms that cause the parasite to become hypersensitive to the antimalarial quinine or the antiviral amantadine. We achieved this by measuring the abilities of these proteins to transport CQ, quinine, and amantadine when expressed in Xenopus oocytes and complemented this work with assays that detect the drug transport activity of PfCRT in its native environment within the parasite. Here we describe two mechanistic explanations for PfCRT-induced drug hypersensitivity. First, we show that quinine, which normally accumulates inside the DV and therewithin exerts its antimalarial effect, binds extremely tightly to the substrate-binding site of certain isoforms of PfCRTCQR. By doing so it likely blocks the normal physiological function of the protein, which is essential for the parasite's survival, and the drug thereby gains an additional killing effect. In the second scenario, we show that although amantadine also sequesters within the DV, the parasite's hypersensitivity to this drug arises from the PfCRTCQR-mediated transport of amantadine from the DV into the cytosol, where it can better access its antimalarial target. In both cases, the mutations that suppress hypersensitivity also abrogate the ability of PfCRTCQR to transport CQ, thus explaining why rescue from hypersensitivity restores the parasite's sensitivity to this antimalarial. These insights provide a foundation for understanding clinically-relevant observations of inverse drug susceptibilities in the malaria parasite.
Asunto(s)
Antimaláricos/farmacología , Resistencia a Medicamentos/fisiología , Malaria Falciparum , Proteínas de Transporte de Membrana/metabolismo , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/metabolismo , Amantadina/metabolismo , Amantadina/farmacología , Animales , Antimaláricos/metabolismo , Transporte Biológico/fisiología , Western Blotting , Cloroquina/metabolismo , Cloroquina/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Mutagénesis Sitio-Dirigida , Isoformas de Proteínas/metabolismo , Quinina/metabolismo , Quinina/farmacología , Xenopus laevisRESUMEN
Organization of the plasma membrane into specialized substructures in different blood lineages facilitates important biological functions including proper localization of receptors at the plasma membrane as well as the initiation of crucial intracellular signaling cascades. The eukaryotic plasma membrane is a lipid bilayer that consists of asymmetrically distributed phospholipids. This asymmetry is actively maintained by membrane-embedded lipid transporters, but there is only limited data available about the molecular identity of the predominantly active transporters and their substrate specificity in different leukocyte subsets. We demonstrate here that the P4-type ATPase ATP11C mediates significant flippase activity in all murine leukocyte subsets. Loss of ATP11C resulted in a defective internalization of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in comparison to control cells. The diminished flippase activity caused increased PS exposure on 7-aminoactinomycin D- (7-AAD-) viable pro-B cells freshly isolated from the bone marrow of ATP11C-deficient mice, which was corrected upon a 2-hour resting period in vitro. Despite the impaired flippase activity in all immune cell subsets, the only other blood cell type with an accumulation of PS on the surface were viable 7-AAD- developing T cells but this did not result in any discernable effect on their development in the thymus. These findings show that all leukocyte lineages exhibit flippase activity, and identify ATP11C as an aminophospholipid translocase in immune cells.
Asunto(s)
Adenosina Trifosfatasas/genética , Linfocitos B/metabolismo , Transporte Biológico Activo/fisiología , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Linfocitos T/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Células Cultivadas , Dactinomicina/análogos & derivados , Dactinomicina/metabolismo , Leucocitos/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: It is becoming increasingly apparent that certain mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) alter the parasite's susceptibility to diverse compounds. Here we investigated the interaction of PfCRT with 3 tricyclic compounds that have been used to treat malaria (quinacrine [QC] and methylene blue [MB]) or to study P. falciparum (acridine orange [AO]). METHODS: We measured the antiplasmodial activities of QC, MB, and AO against chloroquine-resistant and chloroquine-sensitive P. falciparum and determined whether QC and AO affect the accumulation and activity of chloroquine in these parasites. We also assessed the ability of mutant (PfCRT(Dd2)) and wild-type (PfCRT(D10)) variants of the protein to transport QC, MB, and AO when expressed at the surface of Xenopus laevis oocytes. RESULTS: Chloroquine resistance-conferring isoforms of PfCRT reduced the susceptibility of the parasite to QC, MB, and AO. In chloroquine-resistant (but not chloroquine-sensitive) parasites, AO and QC increased the parasite's accumulation of, and susceptibility to, chloroquine. All 3 compounds were shown to bind to PfCRT(Dd2), and the transport of QC and MB via this protein was saturable and inhibited by the chloroquine resistance-reverser verapamil. CONCLUSIONS: Our findings reveal that the PfCRT(Dd2)-mediated transport of tricyclic antimalarials reduces the parasite's susceptibility to these drugs.
Asunto(s)
Proteínas de Transporte de Membrana/metabolismo , Azul de Metileno/metabolismo , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Quinacrina/metabolismo , Verapamilo/farmacología , Animales , Antimaláricos/farmacología , Transporte Biológico/efectos de los fármacos , Resistencia a Medicamentos , Regulación de la Expresión Génica/fisiología , Variación Genética , Oocitos/metabolismo , Xenopus laevisRESUMEN
Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, are the major determinant of chloroquine resistance in this lethal human malaria parasite. Here, we describe P. falciparum lines subjected to selection by amantadine or blasticidin that carry PfCRT mutations (C101F or L272F), causing the development of enlarged food vacuoles. These parasites also have increased sensitivity to chloroquine and some other quinoline antimalarials, but exhibit no or minimal change in sensitivity to artemisinins, when compared with parental strains. A transgenic parasite line expressing the L272F variant of PfCRT confirmed this increased chloroquine sensitivity and enlarged food vacuole phenotype. Furthermore, the introduction of the C101F or L272F mutation into a chloroquine-resistant variant of PfCRT reduced the ability of this protein to transport chloroquine by approximately 93 and 82%, respectively, when expressed in Xenopus oocytes. These data provide, at least in part, a mechanistic explanation for the increased sensitivity of the mutant parasite lines to chloroquine. Taken together, these findings provide new insights into PfCRT function and PfCRT-mediated drug resistance, as well as the food vacuole, which is an important target of many antimalarial drugs.
Asunto(s)
Cloroquina/farmacología , Resistencia a Medicamentos/genética , Proteínas de Transporte de Membrana/genética , Mutación , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/fisiología , Proteínas Protozoarias/genética , Vacuolas/metabolismo , Sustitución de Aminoácidos , Animales , Antimaláricos/farmacología , Transporte Biológico , Humanos , Proteínas de Transporte de Membrana/química , Oocitos/metabolismo , Pruebas de Sensibilidad Parasitaria , Proteínas Protozoarias/química , Xenopus laevisRESUMEN
The intraerythrocytic malaria parasite relies primarily on glycolysis to fuel its rapid growth and reproduction. The major byproduct of this metabolism, lactic acid, is extruded into the external medium. In this study, we show that the human malaria parasite Plasmodium falciparum expresses at its surface a member of the microbial formate-nitrite transporter family (PfFNT), which, when expressed in Xenopus laevis oocytes, transports both formate and lactate. The transport characteristics of PfFNT in oocytes (pH-dependence, inhibitor-sensitivity and kinetics) are similar to those of the transport of lactate and formate across the plasma membrane of mature asexual-stage P. falciparum trophozoites, consistent with PfFNT playing a major role in the efflux of lactate and hence in the energy metabolism of the intraerythrocytic parasite.
Asunto(s)
Formiatos/metabolismo , Ácido Láctico/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Glucólisis , Oocitos/metabolismo , Xenopus laevisRESUMEN
Although efforts to understand the basis for inter-strain phenotypic variation in the most virulent malaria species, Plasmodium falciparum, have benefited from advances in genomic technologies, there have to date been few metabolomic studies of this parasite. Using 1H-NMR spectroscopy, we have compared the metabolite profiles of red blood cells infected with different P. falciparum strains. These included both chloroquine-sensitive and chloroquine-resistant strains, as well as transfectant lines engineered to express different isoforms of the chloroquine-resistance-conferring pfcrt (P. falciparum chloroquine resistance transporter). Our analyses revealed strain-specific differences in a range of metabolites. There was marked variation in the levels of the membrane precursors choline and phosphocholine, with some strains having >30-fold higher choline levels and >5-fold higher phosphocholine levels than others. Chloroquine-resistant strains showed elevated levels of a number of amino acids relative to chloroquine-sensitive strains, including an approximately 2-fold increase in aspartate levels. The elevation in amino acid levels was attributable to mutations in pfcrt. Pfcrt-linked differences in amino acid abundance were confirmed using alternate extraction and detection (HPLC) methods. Mutations acquired to withstand chloroquine exposure therefore give rise to significant biochemical alterations in the parasite.