RESUMO
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.
Assuntos
Formiatos/metabolismo , Ácido Láctico/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Glicólise , Oócitos/metabolismo , Xenopus laevisRESUMO
Mutations in the chloroquine resistance transporter (PfCRT) are the primary determinant of chloroquine (CQ) resistance in the malaria parasite Plasmodium falciparum. A number of distinct PfCRT haplotypes, containing between 4 and 10 mutations, have given rise to CQ resistance in different parts of the world. Here we present a detailed molecular analysis of the number of mutations (and the order of addition) required to confer CQ transport activity upon the PfCRT as well as a kinetic characterization of diverse forms of PfCRT. We measured the ability of more than 100 variants of PfCRT to transport CQ when expressed at the surface of Xenopus laevis oocytes. Multiple mutational pathways led to saturable CQ transport via PfCRT, but these could be separated into two main lineages. Moreover, the attainment of full activity followed a rigid process in which mutations had to be added in a specific order to avoid reductions in CQ transport activity. A minimum of two mutations sufficed for (low) CQ transport activity, and as few as four conferred full activity. The finding that diverse PfCRT variants are all limited in their capacity to transport CQ suggests that resistance could be overcome by reoptimizing the CQ dosage.
Assuntos
Cloroquina/metabolismo , Resistência a Medicamentos , Malária Falciparum/metabolismo , Proteínas de Membrana Transportadoras/genética , Mutação/genética , Parasitos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Transporte Biológico , Haplótipos , Cinética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oócitos , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Transfecção , Xenopus laevisRESUMO
The emergence and spread of chloroquine-resistant Plasmodium falciparum malaria parasites has been a disaster for world health. Resistance is conferred by mutations in the Chloroquine Resistance Transporter (PfCRT), an integral membrane protein localized to the parasite's internal digestive vacuole. These mutations result in a marked reduction in the accumulation of chloroquine (CQ) by the parasite. However, the mechanism by which this occurs is unclear. We expressed both wild-type and resistant forms of PfCRT at the surface of Xenopus laevis oocytes. The resistant form of PfCRT transported CQ, whereas the wild-type protein did not. CQ transport via the mutant PfCRT was inhibited by CQ analogs and by the resistance-reverser verapamil. Thus, CQ resistance is due to direct transport of the drug via mutant PfCRT.
Assuntos
Antimaláricos/metabolismo , Cloroquina/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Antimaláricos/farmacologia , Transporte Biológico/efeitos dos fármacos , Membrana Celular/metabolismo , Cloroquina/análogos & derivados , Cloroquina/farmacologia , Resistência a Medicamentos , Concentração de Íons de Hidrogênio , Potenciais da Membrana , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , Oligopeptídeos/farmacologia , Oócitos/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Verapamil/farmacologia , Xenopus laevisRESUMO
To survive, the human malaria parasite Plasmodium falciparum must acquire pantothenate (vitamin B5) from the external medium. Pantothenol (provitamin B5) inhibits parasite growth by competing with pantothenate for pantothenate kinase, the first enzyme in the coenzyme A biosynthesis pathway. In this study we investigated pantothenol uptake by P. falciparum and in doing so gained insights into the regulation of the parasite's coenzyme A biosynthesis pathway. Pantothenol was shown to enter P. falciparum-infected erythrocytes via two routes, the furosemide-inhibited "new permeation pathways" induced by the parasite in the infected erythrocyte membrane (the sole access route for pantothenate) and a second, furosemide-insensitive pathway. Having entered the erythrocyte, pantothenol is taken up by the intracellular parasite via a mechanism showing functional characteristics distinct from those of the parasite's pantothenate uptake mechanism. On reaching the parasite cytosol, pantothenol is phosphorylated and thereby trapped by pantothenate kinase, shown here to be under feedback inhibition control by coenzyme A. Furosemide reduced this inherent feedback inhibition by competing with coenzyme A for binding to pantothenate kinase, thereby increasing pantothenol uptake.