Characterization of the Commercially-Available Fluorescent Chloroquine-BODIPY Conjugate,LynxTag-CQGREEN,as a Marker for Chloroquine Resistance and Uptake in a 96-Well Plate Assay

Chloroquine was a cheap, extremely effective drug against Plasmodium falciparum until resistance arose. One approach to reversing resistance is the inhibition of chloroquine efflux from its site of action, the parasite digestive vacuole. Chloroquine accumulation studies have traditionally relied on radiolabelled chloroquine, which poses several challenges. There is a need for development of a safe and biologically relevant substitute. We report here a commercially-available green fluorescent chloroquine-BODIPY conjugate, LynxTag-CQ_GREEN, as a proxy for chloroquine accumulation. This compound localized to the digestive vacuole of the parasite as observed under confocal microscopy, and inhibited growth of chloroquine-sensitive strain 3D7 more extensively than in the resistant strains 7G8 and K1. Microplate reader measurements indicated suppression of LynxTag-CQ_GREEN efflux after pretreatment of parasites with known reversal agents. Microsomes carrying either sensitive- or resistant-type PfCRT were assayed for uptake; resistant-type PfCRT exhibited increased accumulation of LynxTag-CQ_GREEN, which was suppressed by pretreatment with known chemosensitizers. Eight laboratory strains and twelve clinical isolates were sequenced for PfCRT and Pgh1 haplotypes previously reported to contribute to drug resistance, and pfmdr1 copy number and chloroquine IC₅₀s were determined. These data were compared with LynxTag-CQ_GREEN uptake/fluorescence by multiple linear regression to identify genetic correlates of uptake. Uptake of the compound correlated with the logIC₅₀ of chloroquine and, more weakly, a mutation in Pgh1, F1226Y.     

Mechanistic basis for multidrug resistance and collateral drug sensitivity conferred to the malaria parasite by polymorphisms in PfMDR1 and PfCRT

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.     

Efflux of a range of antimalarial drugs and ‘chloroquine resistance reversers’ from the digestive vacuole in malaria parasites with mutant PfCRT

Chloroquine‐resistant malaria parasites (Plasmodium falciparum) show an increased leak of H⁺ ions from their internal digestive vacuole in the presence of chloroquine. This phenomenon has been attributed to the transport of chloroquine, together with H⁺, out of the digestive vacuole (and hence away from its site of action) via a mutant form of the parasite's chloroquine resistance transporter (PfCRT). Here, using transfectant parasite lines, we show that a range of other antimalarial drugs, as well as various ‘chloroquine resistance reversers’ induce an increased leak of H⁺ from the digestive vacuole of parasites expressing mutant PfCRT, consistent with these compounds being substrates for mutant forms, but not the wild‐type form, of PfCRT. For some compounds there were significant differences observed between parasites having the African/Asian Dd2 form of PfCRT and those with the South American 7G8 form of PfCRT, consistent with there being differences in the transport properties of the two mutant proteins. The finding that chloroquine resistance reversers are substrates for mutant PfCRT has implications for the mechanism of action of this class of compound.     

Defining the role of PfCRT in Plasmodium falciparum chloroquine resistance

Recent studies have highlighted the importance of a parasite protein referred to as the chloroquine resistance transporter (PfCRT) in the molecular basis of Plasmodium falciparum resistance to the quinoline antimalarials. PfCRT, an integral membrane protein with 10 predicted transmembrane domains, is a member of the drug/metabolite transporter superfamily and is located on the membrane of the intra-erythrocytic parasite's digestive vacuole. Specific polymorphisms in PfCRT are tightly correlated with chloroquine resistance. Transfection studies have now proven that pfcrt mutations confer verapamil-reversible chloroquine resistance in vitro and reveal their important role in resistance to quinine. Available evidence is consistent with the view that PfCRT functions as a transporter directly mediating the efflux of chloroquine from the digestive vacuole.     

Multiple Drugs Compete for Transport via the Plasmodium falciparum Chloroquine Resistance Transporter at Distinct but Interdependent Sites

Mutations in the “chloroquine resistance transporter” (PfCRT) are a major determinant of drug resistance in the malaria parasite Plasmodium falciparum. We have previously shown that mutant PfCRT transports the antimalarial drug chloroquine away from its target, whereas the wild-type form of PfCRT does not. However, little is understood about the transport of other drugs via PfCRT or the mechanism by which PfCRT recognizes different substrates. Here we show that mutant PfCRT also transports quinine, quinidine, and verapamil, indicating that the protein behaves as a multidrug resistance carrier. Detailed kinetic analyses revealed that chloroquine and quinine compete for transport via PfCRT in a manner that is consistent with mixed-type inhibition. Moreover, our analyses suggest that PfCRT accepts chloroquine and quinine at distinct but antagonistically interacting sites. We also found verapamil to be a partial mixed-type inhibitor of chloroquine transport via PfCRT, further supporting the idea that PfCRT possesses multiple substrate-binding sites. Our findings provide new mechanistic insights into the workings of PfCRT, which could be exploited to design potent inhibitors of this key mediator of drug resistance.     

Trafficking of the Phosphoprotein PfCRT to the Digestive Vacuolar Membrane in Plasmodium falciparum

The digestive vacuole plays an important role in the pathophysiology of the human malaria parasite Plasmodium falciparum. It is a terminal degradation organelle involved in the proteolysis of the host erythrocyte's haemoglobin; it is the site of action of several antimalarial drugs and its membrane harbours transporters implicated in drug resistance. How the digestive vacuole recruits residential proteins is largely unknown. Here, we have investigated the mechanism underpinning trafficking of the chloroquine resistance transporter, PfCRT, to the digestive vacuolar membrane. Nested deletion analysis and site‐directed mutagenesis identified threonine 416 as a functional residue for sorting PfCRT to its site of residence. Mass spectroscopy demonstrated that threonine 416 can be phosphorylated. Further phosphorylation was detected at serine 411. Our data establish PfCRT as a phosphoprotein and suggest that phosphorylation of threonine 416 is a possible deciding signal for the sorting of PfCRT to the digestive vacuolar membrane.     

On the mechanism of chloroquine resistance in Plasmodium falciparum

Resistance to chloroquine of malaria strains is known to be associated with a parasite protein named PfCRT, the mutated form of which is able to reduce chloroquine accumulation in the digestive vacuole of the pathogen. Whether the protein mediates extrusion of the drug acting as a channel or as a carrier and which is the protonation state of its chloroquine substrate is the subject of a scientific debate. We present here an analytical approach that explores which combination of hypotheses on the mechanism of transport and the protonation state of chloroquine are consistent with available equilibrium experimental data. We show that the available experimental data are not, by themselves, sufficient to conclude whether the protein acts as a channel or as a transporter, which explains the origin of their different interpretation by different authors. Interestingly, though, each of the two models is only consistent with a subset of hypotheses on the protonation state of the transported molecule. The combination of these results with a sequence and structure analysis of PfCRT, which strongly suggests that the molecule is a carrier, indicates that the transported species is either or both the mono and di-protonated forms of chloroquine. We believe that our results, besides shedding light on the mechanism of chloroquine resistance in P. falciparum, have implications for the development of novel therapies against resistant malaria strains and demonstrate the usefulness of an approach combining systems biology strategies with structural bioinformatics and experimental data.     

Sequence signatures involved in targeting the male-specific lethal complex to X-chromosomal genes in Drosophila melanogaster

Background

Drug resistance in the malaria parasite Plasmodium falciparum severely compromises the treatment and control of malaria. A knowledge of the critical mutations conferring resistance to particular drugs is important in understanding modes of drug action and mechanisms of resistances. They are required to design better therapies and limit drug resistance. A mutation in the gene (pfcrt) encoding a membrane transporter has been identified as a principal determinant of chloroquine resistance in P. falciparum, but we lack a full account of higher level chloroquine resistance. Furthermore, the determinants of resistance in the other major human malaria parasite, P. vivax, are not known. To address these questions, we investigated the genetic basis of chloroquine resistance in an isogenic lineage of rodent malaria parasite P. chabaudi in which high level resistance to chloroquine has been progressively selected under laboratory conditions.

Results

Loci containing the critical genes were mapped by Linkage Group Selection, using a genetic cross between the high-level chloroquine-resistant mutant and a genetically distinct sensitive strain. A novel high-resolution quantitative whole-genome re-sequencing approach was used to reveal three regions of selection on chr11, chr03 and chr02 that appear progressively at increasing drug doses on three chromosomes. Whole-genome sequencing of the chloroquine-resistant parent identified just four point mutations in different genes on these chromosomes. Three mutations are located at the foci of the selection valleys and are therefore predicted to confer different levels of chloroquine resistance. The critical mutation conferring the first level of chloroquine resistance is found in aat1, a putative aminoacid transporter.

Conclusions

Quantitative trait loci conferring selectable phenotypes, such as drug resistance, can be mapped directly using progressive genome-wide linkage group selection. Quantitative genome-wide short-read genome resequencing can be used to reveal these signatures of drug selection at high resolution. The identities of three genes (and mutations within them) conferring different levels of chloroquine resistance generate insights regarding the genetic architecture and mechanisms of resistance to chloroquine and other drugs. Importantly, their orthologues may now be evaluated for critical or accessory roles in chloroquine resistance in human malarias P. vivax and P. falciparum.     

Dictyostelium discoideum expresses a malaria chloroquine resistance mechanism upon transfection with mutant, but not wild-type, Plasmodium falciparum transporter PfCRT

Chloroquine resistance in Plasmodium falciparum malaria results from mutations in PfCRT, a member of a unique family of transporters present in apicomplexan parasites and Dictyostelium discoideum. Mechanisms that have been proposed to explain chloroquine resistance are difficult to evaluate within malaria parasites. Here we report on the targeted expression of wild-type and mutant forms of PfCRT to acidic vesicles in D. discoideum. We show that wild-type PfCRT has minimal effect on the accumulation of chloroquine by D. discoideum, whereas forms of PfCRT carrying a key charge-loss mutation of lysine 76 (e.g. K76T) enable D. discoideum to expel chloroquine. As in P. falciparum, the chloroquine resistance phenotype conferred on transformed D. discoideum can be reversed by the channel-blocking agent verapamil. Although intravesicular pH levels in D. discoideum show small acidic changes with the expression of different forms of PfCRT, these changes would tend to promote intravesicular trapping of chloroquine (a weak base) and do not account for reduced drug accumulation in transformed D. discoideum. Our results instead support outward-directed chloroquine efflux for the mechanism of chloroquine resistance by mutant PfCRT. This mechanism shows structural specificity as D. discoideum transformants that expel chloroquine do not expel piperaquine, a bisquinoline analog of chloroquine used frequently against chloroquine-resistant parasites in Southeast Asia. PfCRT, nevertheless, may have some ability to act on quinine and quinidine. Transformed D. discoideum will be useful for further studies of the chloroquine resistance mechanism and may assist in the development and evaluation of new antimalarial drugs.     

Balancing drug resistance and growth rates via compensatory mutations in the Plasmodium falciparum chloroquine resistance transporter

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.     

PfCRT and the trans-vacuolar proton electrochemical gradient: regulating the access of chloroquine to ferriprotoporphyrin IX

It is accepted that resistance of Plasmodium falciparum to chloroquine (CQ) is caused primarily by mutations in the pfcrt gene. However, a consensus has not yet been reached on the mechanism by which resistance is achieved. CQ-resistant (CQR) parasite lines accumulate less CQ than do CQ-sensitive (CQS) parasites. The CQR phenotype is complex with a component of reduced energy-dependent CQ uptake and an additional component that resembles energy-dependent CQ efflux. Here we show that the required energy input is in the form of the proton electrochemical gradient across the digestive vacuole (DV) membrane. Collapsing the DV proton gradient (or starving the parasites of glucose) results in similar levels of CQ accumulation in CQS and CQR lines. Under these conditions the accumulation of CQ is stimulated in CQR parasite lines but is reduced in CQS lines. Energy deprivation has no effect on the rate of CQ efflux from CQR lines implying that mutant PfCRT does not function as an efflux pump or active carrier. Using pfcrt-modified parasite lines we show that the entire CQ susceptibility phenotype is switched by the single K76T amino acid change in PfCRT. The efflux of CQ in CQR lines is not directly coupled to the energy supply, consistent with a model in which mutant PfCRT functions as a gated channel or pore, allowing charged CQ species to leak out of the DV.     

Assessment of the efficacy of antimalarial drugs recommended by the National Malaria Control Programme in Madagascar: Up-dated baseline data from randomized and multi-site clinical trials

Background

In order to improve the monitoring of the antimalarial drug resistance in Madagascar, a new national network based on eight sentinel sites was set up. In 2006/2007, a multi-site randomized clinical trial was designed to assess the therapeutic efficacy of chloroquine (CQ), sulphadoxine-pyrimethamine (SP), amodiaquine (AQ) and artesunate plus amodiaquine combination (ASAQ), the antimalarial therapies recommended by the National Malaria Control Programme (NMCP).

Methods

Children between six months and 15 years of age, with uncomplicated falciparum malaria, were enrolled. Primary endpoints were the day-14 and day-28 risks of parasitological failure, either unadjusted or adjusted by genotyping. Risks of clinical and parasitological treatment failure after adjustment by genotyping were estimated using Kaplan-Meier survival analysis. Secondary outcomes included fever clearance, parasite clearance, change in haemoglobin levels between Day 0 and the last day of follow-up, and the incidence of adverse events.

Results

A total of 1,347 of 1,434 patients (93.9%) completed treatment and follow-up to day 28. All treatment regimens, except for the chloroquine (CQ) treatment group, resulted in clinical cure rates above 97.6% by day-14 and 96.7% by day-28 (adjusted by genotyping). Parasite and fever clearance was more rapid with artesunate plus amodiaquine, but the extent of haematological recovery on day-28 did not differ significantly between the four groups. No severe side-effects were observed during the follow-up period.

Conclusion

These findings (i) constitute an up-dated baseline data on the efficacy of antimalarial drugs recommended by the NMCP, (ii) show that antimalarial drug resistance remains low in Madagascar, except for CQ, compared to the bordering countries in the Indian Ocean region such as the Comoros Archipelago and (iii) support the current policy of ASAQ as the first-line treatment in uncomplicated falciparum malaria.

     

Deployment of ACT antimalarials for treatment of malaria: challenges and opportunities

Background

In late 2002, the health authorities of Mozambique implemented sulphadoxine-pyrimethamine (SP)/amodiaquine (AQ) as first-line treatment against uncomplicated falciparum malaria. In 2004, this has been altered to SP/artesunate in line with WHO recommendations of using Artemisinin Combination Therapies (ACTs), despite the fact that all the neighbouring countries have abandoned SP-drug combinations due to high levels of SP drug resistance. In the study area, one year prior to the change to SP/AQ, SP alone was used to treat uncomplicated malaria cases. The study described here investigated the immediate impact of the change to SP on the frequency of SP and CQ resistance-related haplotypes in the Plasmodium falciparum genes Pfdhfr, Pfdhps and Pfcrt before and a year after the introduction of SP.

Methods

Samples were collected during two cross sectional surveys in early 2002 and 2003 involving 796 and 692 children one year or older and adults randomly selected living in Maciana, an area located in Manhiça district, Southern Mozambique. Out of these, 171 and 173 P. falciparum positive samples were randomly selected to measure the frequency of resistance- related haplotypes in Pfdhfr, Pfdhps and Pfcrt based on results obtained by nested PCR followed by sequence-specific oligonucleotide probe (SSOP)-ELISA.

Results

The frequency of the SP-resistance associated Pfdhps double mutant (SGEAA) haplotype increased significantly from 14% to 35% (P < 0.001), while the triple mutant Pfdhfr haplotype (CIRNI) remained high and only changed marginally from 46% to 53% (P = 0.405) after one year with SP as first-line treatment in the study area. Conversely, the combined Pfdhfr/Pfdhps quintuple mutant haplotype increased from 8% to 26% (P = 0.005). The frequency of the chloroquine resistance associated Pfcrt -CVIET haplotype was above 90% in both years.

Conclusion

These retrospective findings add to the general concern on the lifespan of the combination of SP/artesunate in Mozambique. The high frequency of quintuple Pfdhfr/Pfdhps haplotypes found here as early as 2002 most likely cause ideal conditions for the development of artesunate tolerance in the P. falciparum populations and may even endanger the sensitivity to the second-line drug, Coartem^®.     

The cardiovascular effects of amodiaquine and structurally related antimalarials: An individual patient data meta-analysis

BackgroundAmodiaquine is a 4-aminoquinoline antimalarial similar to chloroquine that is used extensively for the treatment and prevention of malaria. Data on the cardiovascular effects of amodiaquine are scarce, although transient effects on cardiac electrophysiology (electrocardiographic QT interval prolongation and sinus bradycardia) have been observed. We conducted an individual patient data meta-analysis to characterise the cardiovascular effects of amodiaquine and thereby support development of risk minimisation measures to improve the safety of this important antimalarial.Methods and findingsStudies of amodiaquine for the treatment or prevention of malaria were identified from a systematic review. Heart rates and QT intervals with study-specific heart rate correction (QTcS) were compared within studies and individual patient data pooled for multivariable linear mixed effects regression.The meta-analysis included 2,681 patients from 4 randomised controlled trials evaluating artemisinin-based combination therapies (ACTs) containing amodiaquine (n = 725), lumefantrine (n = 499), piperaquine (n = 716), and pyronaridine (n = 566), as well as monotherapy with chloroquine (n = 175) for uncomplicated malaria. Amodiaquine prolonged QTcS (mean = 16.9 ms, 95% CI: 15.0 to 18.8) less than chloroquine (21.9 ms, 18.3 to 25.6, p = 0.0069) and piperaquine (19.2 ms, 15.8 to 20.5, p = 0.0495), but more than lumefantrine (5.6 ms, 2.9 to 8.2, p < 0.001) and pyronaridine (−1.2 ms, −3.6 to +1.3, p < 0.001). In individuals aged ≥12 years, amodiaquine reduced heart rate (mean reduction = 15.2 beats per minute [bpm], 95% CI: 13.4 to 17.0) more than piperaquine (10.5 bpm, 7.7 to 13.3, p = 0.0013), lumefantrine (9.3 bpm, 6.4 to 12.2, p < 0.001), pyronaridine (6.6 bpm, 4.0 to 9.3, p < 0.001), and chloroquine (5.9 bpm, 3.2 to 8.5, p < 0.001) and was associated with a higher risk of potentially symptomatic sinus bradycardia (≤50 bpm) than lumefantrine (risk difference: 14.8%, 95% CI: 5.4 to 24.3, p = 0.0021) and chloroquine (risk difference: 8.0%, 95% CI: 4.0 to 12.0, p < 0.001). The effect of amodiaquine on the heart rate of children aged <12 years compared with other antimalarials was not clinically significant. Study limitations include the unavailability of individual patient-level adverse event data for most included participants, but no serious complications were documented.ConclusionsWhile caution is advised in the use of amodiaquine in patients aged ≥12 years with concomitant use of heart rate–reducing medications, serious cardiac conduction disorders, or risk factors for torsade de pointes, there have been no serious cardiovascular events reported after amodiaquine in widespread use over 7 decades. Amodiaquine and structurally related antimalarials in the World Health Organization (WHO)-recommended dose regimens alone or in ACTs are safe for the treatment and prevention of malaria.

In this meta-analysis, Xin Hui Supanee Chan and colleagues investigate the cardiovascular effects of amodiaquine and structurally-related antimalarials using individual patient data from trials.     

Functional reconstitution of purified chloroquine resistance membrane transporter expressed in yeast

Malaria is one of the major parasitic diseases. Current treatment of malaria is seriously hampered by the emergence of drug resistant cases. A once-effective drug chloroquine (CQ) has been rendered almost useless. The mechanism of CQ resistance is complicated and largely unknown. Recently, a novel transmembrane protein, Plasmodium falciparum chloroquine resistance transporter (PfCRT), has fulfilled all the requirements of being the CQ resistance gene. In order to elucidate the mechanism how PfCRT mediates CQ resistance, we have cloned the cDNA from a CQ sensitive parasite (3D7) and tried to express it in Pichia pastoris (P. pastoris) but with unsuccessful results due to AT-rich sequences in the malaria genome. We have therefore, based on the codon usage in P. pastoris, chemically synthesized a codon-modified pfcrt with an overall 55% AT content. This codon-modified pfcrt has now been successfully expressed in P. pastoris. The expressed PfCRT has been purified with immuno metal affinity chromatography (IMAC) and then reconstituted into proteoliposome. It was found that proteoliposomes have a saturable, concentration and time-dependent CQ transport activity. In addition, we found that proteoliposomes with resistant PfCRT(r) (K76T or K76I) showed an increased CQ transport activity compared to liposomes with lipid alone, or proteoliposomes reconstituted with sensitive PfCRT(s) (K76) protein. This activity could be inhibited by nigericin and decreased with the removal of Cl(-). This work suggests that PfCRT is mediating CQR in P. falciparum by virtue of its changes in CQ transport activity depending on pH gradient and chloride ion in the food vacuole.     

Genetic linkage analyses redefine the roles of PfCRT and PfMDR1 in drug accumulation and susceptibility in Plasmodium falciparum

Resistance to quinoline antimalarial drugs has emerged in different parts of the world and involves sets of discrete mutational changes in pfcrt and pfmdr1 in the human malaria parasite Plasmodium falciparum. To better understand how the different polymorphic haplotypes of pfmdr1 and pfcrt contribute to drug resistance, we have conducted a linkage analysis in the F1 progeny of a genetic cross where we assess both the susceptibility and the amount of accumulation of chloroquine, amodiaquine, quinine and quinidine. Our data show that the different pfcrt and pfmdr1 haplotypes confer drug-specific responses which, depending on the drug, may affect drug accumulation or susceptibility or both. These findings suggest that PfCRT and PfMDR1 are carriers of antimalarial drugs, but that the interaction with a drug interferes with the carriers' natural transport function such that they are now themselves targets of these drugs. How well a mutant PfCRT and PfMDR1 type copes with its competing transport functions is determined by its specific sets of amino acid substitutions.     

Characterization of the Chloroquine Resistance Transporter Homologue in Toxoplasma gondii

Mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein confer resistance to the antimalarial drug chloroquine. PfCRT localizes to the parasite digestive vacuole, the site of chloroquine action, where it mediates resistance by transporting chloroquine out of the digestive vacuole. PfCRT belongs to a family of transporter proteins called the chloroquine resistance transporter family. CRT family proteins are found throughout the Apicomplexa, in some protists, and in plants. Despite the importance of PfCRT in drug resistance, little is known about the evolution or native function of CRT proteins. The apicomplexan parasite Toxoplasma gondii contains one CRT family protein. We demonstrate that T. gondii CRT (TgCRT) colocalizes with markers for the vacuolar (VAC) compartment in these parasites. The TgCRT-containing VAC is a highly dynamic organelle, changing its morphology and protein composition between intracellular and extracellular forms of the parasite. Regulated knockdown of TgCRT expression resulted in modest reduction in parasite fitness and swelling of the VAC, indicating that TgCRT contributes to parasite growth and VAC physiology. Together, our findings provide new information on the role of CRT family proteins in apicomplexan parasites.     

A Genome Wide Association Study of Plasmodium falciparum Susceptibility to 22 Antimalarial Drugs in Kenya

Background

Drug resistance remains a chief concern for malaria control. In order to determine the genetic markers of drug resistant parasites, we tested the genome-wide associations (GWA) of sequence-based genotypes from 35 Kenyan P. falciparum parasites with the activities of 22 antimalarial drugs.

Methods and Principal Findings

Parasites isolated from children with acute febrile malaria were adapted to culture, and sensitivity was determined by in vitro growth in the presence of anti-malarial drugs. Parasites were genotyped using whole genome sequencing techniques. Associations between 6250 single nucleotide polymorphisms (SNPs) and resistance to individual anti-malarial agents were determined, with false discovery rate adjustment for multiple hypothesis testing. We identified expected associations in the pfcrt region with chloroquine (CQ) activity, and other novel loci associated with amodiaquine, quinazoline, and quinine activities. Signals for CQ and primaquine (PQ) overlap in and around pfcrt, and interestingly the phenotypes are inversely related for these two drugs. We catalog the variation in dhfr, dhps, mdr1, nhe, and crt, including novel SNPs, and confirm the presence of a dhfr-164L quadruple mutant in coastal Kenya. Mutations implicated in sulfadoxine-pyrimethamine resistance are at or near fixation in this sample set.

Conclusions/Significance

Sequence-based GWA studies are powerful tools for phenotypic association tests. Using this approach on falciparum parasites from coastal Kenya we identified known and previously unreported genes associated with phenotypic resistance to anti-malarial drugs, and observe in high-resolution haplotype visualizations a possible signature of an inverse selective relationship between CQ and PQ.     

Submicroscopic Gametocytes and the Transmission of Antifolate-Resistant Plasmodium falciparum in Western Kenya

Background

Single nucleotide polymorphisms (SNPs) in the dhfr and dhps genes are associated with sulphadoxine-pyrimethamine (SP) treatment failure and gametocyte carriage. This may result in enhanced transmission of mutant malaria parasites, as previously shown for chloroquine resistant parasites. In the present study, we determine the association between parasite mutations, submicroscopic P. falciparum gametocytemia and malaria transmission to mosquitoes.

Methodology/Principal Findings

Samples from children treated with SP alone or in combination with artesunate (AS) or amodiaquine were genotyped for SNPs in the dhfr and dhps genes. Gametocytemia was determined by microscopy and Pfs25 RNA–based quantitative nucleic acid sequence–based amplification (Pfs25 QT-NASBA). Transmission was determined by membrane-feeding assays. We observed no wild type infections, 66.5% (127/191) of the infections expressed mutations at all three dhfr codons prior to treatment. The presence of all three mutations was not related to higher Pfs25 QT-NASBA gametocyte prevalence or density during follow-up, compared to double mutant infections. The proportion of infected mosquitoes or oocyst burden was also not related to the number of mutations. Addition of AS to SP reduced gametocytemia and malaria transmission during follow-up.

Conclusions/Significance

In our study population where all infections had at least a double mutation in the dhfr gene, additional mutations were not related to increased submicroscopic gametocytemia or enhanced malaria transmission. The absence of wild-type infections is likely to have reduced our power to detect differences. Our data further support the use of ACT to reduce the transmission of drug-resistant malaria parasites.