Plasmodium falciparum chloroquine resistance transporter is a H+-coupled polyspecific nutrient and drug exporter.
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ABSTRACT:
SUBMITTER: Juge N
PROVIDER: S-EPMC4371956 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The widespread use of chloroquine to treat Plasmodium falciparum infections has resulted in the selection and dissemination of variant haplotypes of the primary resistance determinant PfCRT. These haplotypes have encountered drug pressure and within-host competition with wild-type drug-sensitive parasites. To examine these selective forces in vitro, we genetically engineered P.?falciparum to express geographically diverse PfCRT haplotypes. Variant alleles from the Philippines (PH1 and PH2, which differ solely by the C72S mutation) both conferred a moderate gain of chloroquine resistance and a reduction in growth rates in vitro. Of the two, PH2 showed higher IC50 values, contrasting with reduced growth. Furthermore, a highly mutated pfcrt allele from Cambodia (Cam734) conferred moderate chloroquine resistance and enhanced growth rates, when tested against wild-type pfcrt in co-culture competition assays. These three alleles mediated cross-resistance to amodiaquine, an antimalarial drug widely used in Africa. Each allele, along with the globally prevalent Dd2 and 7G8 alleles, rendered parasites more susceptible to lumefantrine, the partner drug used in the leading first-line artemisinin-based combination therapy. These data reveal ongoing region-specific evolution of PfCRT that impacts drug susceptibility and relative fitness in settings of mixed infections, and raise important considerations about optimal agents to treat chloroquine-resistant malaria.
| S-EPMC4511469 | biostudies-literature
Project description:Several models describing how amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer resistance to chloroquine (CQ) and other antimalarial drugs have been proposed. Further progress requires molecular analysis of interactions between purified reconstituted PfCRT protein and these drugs. We have thus designed and synthesized several perfluorophenyl azido (pfpa) CQ analogues for PfCRT photolabeling studies. One particularly useful probe (AzBCQ) places the pfpa group at the terminal aliphatic N of CQ via a flexible four-carbon ester linker and includes a convenient biotin tag. This probe photolabels PfCRT in situ with high specificity. Using reconstituted proteoliposomes harboring partially purified recombinant PfCRT, we analyze AzBCQ photolabeling versus competition with CQ and other drugs to probe the nature of the CQ binding site. We also inspect how pH, the chemoreversal agent verapamil (VPL), and various amino acid mutations in PfCRT that cause CQ resistance (CQR) affect the efficiency of AzBCQ photolabeling. Upon gel isolation of AzBCQ-labeled PfCRT followed by trypsin digestion and mass spectrometry analysis, we are able to define a single AzBCQ covalent attachment site lying within the digestive vacuolar-disposed loop between putative helices 9 and 10 of PfCRT. Taken together, the data provide important new insight into PfCRT function and, along with previous results, allow us to propose a model for a single CQ binding site in the PfCRT protein.
| S-EPMC2724749 | biostudies-literature
Project description:Mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer resistance to several antimalarial drugs such as chloroquine (CQ) or piperaquine (PPQ), a partner molecule in current artemisinin-based combination therapies. As a member of the Drug/Metabolite Transporter (DMT) superfamily, the vacuolar transporter PfCRT may translocate substrate molecule(s) across the membrane of the digestive vacuole (DV), a lysosome-like organelle. However, the physiological substrate(s), the transport mechanism and the functional regions of PfCRT remain to be fully characterized. Here, we hypothesized that identification of evolutionary conserved sites in a tertiary structural context could help locate putative functional regions of PfCRT. Hence, site-specific substitution rates were estimated over Plasmodium evolution at each amino acid sites, and the PfCRT tertiary structure was predicted in both inward-facing (open-to-vacuole) and occluded states through homology modeling using DMT template structures sharing <15% sequence identity with PfCRT. We found that the vacuolar-half and membrane-spanning domain (and especially the transmembrane helix 9) of PfCRT were more conserved, supporting that its physiological substrate is expelled out of the parasite DV. In the PfCRT occluded state, some evolutionary conserved sites, including positions related to drug resistance mutations, participate in a putative binding pocket located at the core of the PfCRT membrane-spanning domain. Through structural comparison with experimentally-characterized DMT transporters, we identified several conserved PfCRT amino acid sites located in this pocket as robust candidates for mediating substrate transport. Finally, in silico mutagenesis revealed that drug resistance mutations caused drastic changes in the electrostatic potential of the transporter vacuolar entry and pocket, facilitating the escape of protonated CQ and PPQ from the parasite DV.
| S-EPMC7076037 | biostudies-literature
Project description:The chloroquine resistance transporter PfCRT of the human malaria parasite Plasmodium falciparum confers resistance to the former first-line antimalarial drug chloroquine, and it modulates the responsiveness to a wide range of quinoline and quinoline-like compounds. PfCRT is post-translationally modified by phosphorylation, palmitoylation, and, possibly, ubiquitination. However, the impact of these post-translational modifications on P. falciparum biology and, in particular, the drug resistance-conferring activity of PfCRT has remained elusive. Here, we confirm phosphorylation at Ser-33 and Ser-411 of PfCRT of the chloroquine-resistant P. falciparum strain Dd2 and show that kinase inhibitors can sensitize drug responsiveness. Using CRISPR/Cas9 genome editing to generate genetically engineered PfCRT variants in the parasite, we further show that substituting Ser-33 with alanine reduced chloroquine and quinine resistance by ?50% compared with the parental P. falciparum strain Dd2, whereas the phosphomimetic amino acid aspartic acid could fully and glutamic acid could partially reconstitute the level of chloroquine/quinine resistance. Transport studies conducted in the parasite and in PfCRT-expressing Xenopus laevis oocytes linked phosphomimetic substitution at Ser-33 to increased transport velocity. Our data are consistent with phosphorylation of Ser-33 relieving an autoinhibitory intramolecular interaction within PfCRT, leading to a stimulated drug transport activity. Our findings shed additional light on the function of PfCRT and suggest that chloroquine could be reevaluated as an antimalarial drug by targeting the kinase in P. falciparum that phosphorylates Ser-33 of PfCRT.
| S-EPMC6709616 | biostudies-literature
Project description:BACKGROUND: In Haiti where chloroquine (CQ) is widely used for malaria treatment, reports of resistance are scarce. However, recent identification of CQ resistance genotypes in one site is suggestive of an emerging problem. Additional studies are needed to evaluate genetic mutations associated with CQ resistance, especially in the Plasmodium falciparum multi-drug resistance-1 gene (pfmdr1) while expanding the already available information on P. falciparum CQ transporter gene (pfcrt) in Haiti. METHODS: Blood samples were collected on Whatman filter cards (FTA) from eight clinics spread across Haiti. Following the confirmation of P. falciparum in the samples, PCR protocols were used to amplify regions of pfmdr1and pfcrt codons of interest, (86, 184, 1034, 1042, and 1246) and (72-76), respectively. Sequencing and site-specific restriction enzyme digestions were used to analyse these DNA fragments for the presence of single nucleotide polymorphisms (SNPs) known to confer resistance to anti-malarial drugs. RESULTS: P. falciparum infection was confirmed in160 samples by amplifying a segment of the P. falciparum 18S small subunit ribosomal RNA gene (pfssurrna). The sequence of pfmdr1 in 54 of these samples was determined between codons 86,184 codons 1034, 1042 and 1246. No sequence differences from that of the NF54 clone 3D7 were found among the 54 samples except at codon 184, where a non-silent mutation was found in all samples predicted to alter the amino acid sequence replacing tyrosine with phenylalanine (Y184F). This altered sequence was also confirmed by restriction enzyme digestion. The sequence of pfmdr1 at codons 86, 184, 1034 and 1042 encoded the NFSN haplotype. The sequence of pfcrt codons 72-76 from 79 samples was determined and found to encode CVMNK, consistent with a CQ sensitive genotype. CONCLUSION: The presence of the Y184F mutation in pfmdr1 of P. falciparum parasites in Haiti may have implications for resistance to antimalarial drugs. The absence of mutation in pfcrt at codon 76 among 79 isolates tested suggests that sensitivity to CQ in Haiti remains common. Wide-spread screening of the pfmdr1 and pfcrt especially among patients experiencing treatment failure may be a useful tool in early detection of the emergence of antimalarial drug resistance in Haiti.
| S-EPMC3879102 | biostudies-literature
Project description:Using a recently elucidated atomic-resolution cryogenic electron microscopy (cryo-EM) structure for the Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein 7G8 isoform as template [Kim, J.; Nature 2019, 576, 315-320], we use Monte Carlo molecular dynamics (MC/MD) simulations of PfCRT embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane to solve energy-minimized structures for 7G8 PfCRT and two additional PfCRT isoforms that harbor 5 or 7 amino acid substitutions relative to 7G8 PfCRT. Guided by drug binding previously defined using chloroquine (CQ) photoaffinity probe labeling, we also use MC/MD energy minimization to elucidate likely CQ binding geometries for the three membrane-embedded isoforms. We inventory salt bridges and hydrogen bonds in these structures and summarize how the limited changes in primary sequence subtly perturb local PfCRT isoform structure. In addition, we use the "AlphaFold" artificial intelligence AlphaFold2 (AF2) algorithm to solve for domain structure that was not resolved in the previously reported 7G8 PfCRT cryo-EM structure, and perform MC/MD energy minimization for the membrane-embedded AF2 structures of all three PfCRT isoforms. We compare energy-minimized structures generated using cryo-EM vs AF2 templates. The results suggest how amino acid substitutions in drug resistance-associated isoforms of PfCRT influence PfCRT structure and CQ transport.
| S-EPMC10950298 | biostudies-literature
Project description: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.
| S-EPMC4588581 | biostudies-literature
Project description:The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide1. Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy2. Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum CQ-resistance transporter PfCRT, a 49-kDa member of the drug/metabolite transporter superfamily that traverses the membrane of the acidic digestive vacuole of the parasite3-9. Here we present the structure, at 3.2 Å resolution, of the PfCRT isoform of CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy and antigen-binding fragment technology. Mutations that contribute to CQ and PPQ resistance localize primarily to moderately conserved sites on distinct helices that line a central negatively charged cavity, indicating that this cavity is the principal site of interaction with the positively charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, and that these drugs are mutually competitive. The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance5, but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America, respectively6,9, reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest that distinct mechanistic features mediate the resistance to CQ and PPQ in PfCRT variants. These data provide atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures.
| S-EPMC6911266 | biostudies-literature
Project description:Malaria parasites break down host haemoglobin into peptides and amino acids in the digestive vacuole for export to the parasite cytoplasm for growth: interrupting this process is central to the mode of action of several antimalarial drugs. Mutations in the chloroquine (CQ) resistance transporter, pfcrt, located in the digestive vacuole membrane, confer CQ resistance in Plasmodium falciparum, and typically also affect parasite fitness. However, the role of other parasite loci in the evolution of CQ resistance is unclear. Here we use a combination of population genomics, genetic crosses and gene editing to demonstrate that a second vacuolar transporter plays a key role in both resistance and compensatory evolution. Longitudinal genomic analyses of the Gambian parasites revealed temporal signatures of selection on a putative amino acid transporter (pfaat1) variant S258L, which increased from 0% to 97% in frequency between 1984 and 2014 in parallel with the pfcrt1 K76T variant. Parasite genetic crosses then identified a chromosome 6 quantitative trait locus containing pfaat1 that is selected by CQ treatment. Gene editing demonstrated that pfaat1 S258L potentiates CQ resistance but at a cost of reduced fitness, while pfaat1 F313S, a common southeast Asian polymorphism, reduces CQ resistance while restoring fitness. Our analyses reveal hidden complexity in CQ resistance evolution, suggesting that pfaat1 may underlie regional differences in the dynamics of resistance evolution, and modulate parasite resistance or fitness by manipulating the balance between both amino acid and drug transport.
| S-EPMC10322710 | biostudies-literature
Project description:INTRODUCTION:Malaria is a human disease of which causes high morbidity and mortality. In Plasmodium falciparum malaria, the resistance to antimalarial drugs, especially chloroquine (CQ) is one of the paramount factors contributing to the global increase in morbidity and mortality, due to malaria. Hence, there is a need for detection of chloroquine drug resistance genes i.e., pfcrt-o (Plasmodium falciparum chloroquine resistance transporter-o) and pfmdr-1 (Plasmodium falciparum multidrug resistance-1) of P. falciparum and pvcrt-o (Plasmodium vivax chloroquine resistance transporter-o) and pvmdr-1 (Plasmodium vivax multidrug resistance-1) of P. vivax by using molecular methods to prevent mortality in malarial cases. AIM:To standardize chloroquine drug sensitivity testing by molecular method so as to provide reports of chloroquine within 6-8 hours to physicians for better treatment. MATERIALS AND METHODS:This study was conducted over a period of one year from January to December 2014. A Total of 300 blood samples were collected from malaria suspected patient attending MGM Hospital, Kamothe, Navi Mumbai, India. Out of 300 blood samples, 44 were malaria positive as assessed by Thick and Thin blood smear stained, by Leishman's method and examination with light microscope. Chloroquine drug sensitivity testing was performed using WHO III plate method (micro test). Nested PCR was done for detection of pfcrt-o and pfmdr-1 for P. falciparum and pvcrt-o, pvmdr-1 genes for P. vivax. RESULTS:Total 44 samples were included in this study, out of which 22 samples confirmed for Plasmodium falciparum and 22 samples confirmed for Plasmodium vivax. Out of 22 P. falciparum 15 (68.18%) samples were chloroquine resistant. P. vivax showed chloroquine resistance to 5 samples (22.73%) by method similar to WHO III plate method (micro test) and nested PCR. CONCLUSION:Drug resistance testing by molecular methods is useful for early detection of antimalarial drug resistance. pfmdr-1 along with pfcrt-o can be used as biomarker for chloroquine drug resistance in P. falciparum and pvmdr-1 along with pvcrt-o for P. vivax.
| S-EPMC5020184 | biostudies-literature