54 research outputs found
Exploring the substrate specificity of the Plasmodium falciparum 'chloroquine resistance transporter'
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. The native substrates of PfCRT were investigated in both the Xenopus oocyte expression system and isogenic parasite lines expressing different pfcrt isoforms, using complementary physiological, biochemical, and metabolomic approaches. Host-derived peptides of 4-11 amino acid residues, varying in both charge and composition, were identified as the substrates of PfCRT in vitro and in situ, and the protein was shown not to mediate the non-specific transport of ions and other metabolites. Drug-resistance-conferring mutations were found to reduce both the peptide transport capacity and substrate range of PfCRT, explaining the impaired fitness of drug-resistant parasites. The transport of peptides and peptide mimics via PfCRT is saturable and inhibited by verapamil. The results presented in this Thesis 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 long-awaited resolution of PfCRT's substrate-specificity and physiological role will aid the development of drugs that target PfCRT and/or restore the efficacy of existing antimalarials. It is becoming increasingly apparent that certain mutations in PfCRT alter the parasite's susceptibility to a range of pharmacons. Here, the interactions of PfCRT with a structurally diverse group of compounds - methylene blue, quinacrine, amantadine, and piperaquine - were investigated. Parasite susceptibility to methylene blue, quinacrine, and amantadine against CQ-resistant and CQ-sensitive P. falciparum was measured. Parasites expressing CQ-resistance-conferring isoforms of PfCRT exhibit reduced susceptibility to methylene blue and quinacrine but increased susceptibility to amantadine. The ability of wild-type and mutant isoforms of PfCRT to transport methylene blue, quinacrine, amantadine, and piperaquine when expressed at the surface of Xenopus oocytes was also assessed. Mutant isoforms of PfCRT mediated transport of methylene blue, quinacrine, amantadine, and piperaquine, whereas wild-type PfCRT did not. Furthermore, transport of these four drugs via mutant PfCRT was saturable and inhibited by verapamil. In this Thesis, possible mechanistic explanations for PfCRT-induced drug resistance to methylene blue, quinacrine, and piperaquine and PfCRT-induced drug hypersensitivity to amantadine are described. First, methylene blue, quinacrine, and piperaquine, which normally accumulate inside the digestive vacuole and therewithin exert their antimalarial effects, are transported out of this compartment via mutant isoforms of PfCRT. The second scenario shows that although amantadine also sequesters within the digestive vacuole, the parasite's hypersensitivity to this drug arises from the CQ-resistance-conferring isoforms of PfCRT mediating the transport of amantadine from the digestive vacuole into the cytosol, where it can better access its antimalarial target. These insights provide a foundation for understanding clinically-relevant observations of drug resistance and inverse drug susceptibilities in the malaria parasite
Molecular Mechanisms for Drug Hypersensitivity Induced by the Malaria Parasite's Chloroquine Resistance Transporter
Mutations in the Plasmodium falciparum ‘chloroquine resistance transporter’ (PfCRT) confer resistance to chloroquine (CQ) and related antimalarials by enabling the protein to transportthese 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
A lactate and formate transporter in the intraerythrocytic malaria parasite, Plasmodium falciparum
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.This work was supported by grants from the Australian National Health and Medical
Research Council (NHMRC; 316933 and 525428 to K.K. and 1007035 to R.E.M.), and by
the L’Ore´al Australia For Women in Science programme (R.E.M.). A.M.L. was supported
by an NHMRC Overseas Biomedical Fellowship (585519) and R.E.M. was supported by
NHMRC Australian Biomedical Fellowships (520320 and 1053082)
1H-NMR metabolite profiles of different strains of Plasmodium falciparum
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. Chloroquineresistant 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
Verapamil-Sensitive Transport of Quinacrine and Methylene Blue via the Plasmodium falciparum Chloroquine Resistance Transporter Reduces the Parasite's Susceptibility to these Tricyclic Drugs.
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
The Malaria Parasite's Lactate Transporter PfFNT Is the Target of Antiplasmodial Compounds Identified in Whole Cell Phenotypic Screens
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.Aspects of this work were supported by a
Project Grant (1042272 to KK) from the Australian
National Health and Medical Research Council
(NHMRC). AML is the recipient of an Australian
Research Council Discovery Early Career
Researcher Award (DE160101035), REM was
supported by a NHMRC Career Development Fellowship (1053082) and MJM is a NHMRC
Principal Research Fello
Recommended from our members
Surfactant modulated interaction of hydrophobically modified ethoxylated urethane (HEUR) polymers with impenetrable surfaces
Hypothesis:
The presence of surfactant modulates the surface-chemistry-specific interaction of hard colloidal particles (latex) with HEUR polymers, principally through introducing a preferential solution interaction rather than a competitive surface interaction; addition of surfactant leads to a preponderance of polymer/surfactant solution complexes rather than surface-bound complexes.
Experiments:
A range of model formulations comprising a hexyl end-capped urethane polymer (C6-L-(EO100-L)9-C6), sodium dodecylsulfate (SDS) and a series of polystyrene-butylacrylate latices (PS-BA-L) have been characterised in terms of rheology, particle surface area (solvent relaxation NMR), polymer conformation (small-angle neutron scattering) and solution composition to build up a detailed picture of the distribution of the HEUR in the presence of both surfactant and latex.
Findings:
There is very weak adsorption of C6-L-(EO100-L)9-C6 to only the most hydrophobic latex surface studied, an adsorption that is further weakened by the addition of low levels of surfactant. Macroscopic changes in the hydrophobic latex system may be interpreted in terms of bridging flocculation at low polymer concentrations. No adsorption of C6-L-(EO100-L)9-C6 is observed in the case of hydrophilic surfaces. In most cases, the observed behaviour of the ternary system (polymer/surfactant/particle) is highly reminiscent of the binary (polymer/surfactant) system at the appropriate composition, suggesting that the polymer/surfactant solution interaction is the dominant one
Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, enlarge the parasite's food vacuole and alter drug sensitivities
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
TFOS:Unique challenges and unmet needs for the management of ocular surface diseases throughout the world
Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, enlarge the parasite's food vacuole and alter drug sensitivities OPEN
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
- …