RESUMEN
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
Dopamine is an important neuromodulator involved in reward-processing, movement control, motivational responses, and other aspects of behavior in most animals. In honey bees (Apis mellifera), the dopaminergic system has been implicated in an elaborate pheromonal communication network between individuals and in the differentiation of females into reproductive (queen) and sterile (worker) castes. Here we have identified and characterized a honey bee dopamine transporter (AmDAT) and a splice variant lacking exon 3 (AmDATΔex3). Both transcripts are present in the adult brain and antennae as well as at lower levels within larvae and ovaries. When expressed separately in the Xenopus oocyte system, AmDAT localizes to the oocyte surface whereas the splice variant is retained at an internal membrane. Oocytes expressing AmDAT exhibit a 12-fold increase in the uptake of [3H]dopamine relative to non-injected oocytes, whereas the AmDATΔex3-expressing oocytes show no change in [3H]dopamine transport. Electrophysiological measurements of AmDAT activity revealed it to be a high-affinity, low-capacity transporter of dopamine. The transporter also recognizes noradrenaline as a major substrate and tyramine as a minor substrate, but does not transport octopamine, L-Dopa, or serotonin. Dopamine transport via AmDAT is inhibited by cocaine in a reversible manner, but is unaffected by octopamine. Co-expression of AmDAT and AmDATΔex3 in oocytes results in a substantial reduction in AmDAT-mediated transport, which was also detected as a significant decrease in the level of AmDAT protein. This down-regulatory effect is not attributable to competition with AmDATΔex3 for ER ribosomes, nor to a general inhibition of the oocyte's translational machinery. In vivo, the expression of both transcripts shows a high level of inter-individual variability. Gene-focused, ultra-deep amplicon sequencing detected methylation of the amdat locus at ten 5'-C-phosphate-G-3' dinucleotides (CpGs), but only in 5-10% of all reads in whole brains or antennae. These observations, together with the localization of the amdat transcript to a few clusters of dopaminergic neurons, imply that amdat methylation is positively linked to its transcription. Our findings suggest that multiple cellular mechanisms, including gene splicing and epigenomic communication systems, may be adopted to increase the potential of a conserved gene to contribute to lineage-specific behavioral outcomes.
RESUMEN
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
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
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.