Search

Over the last few decades, malaria-derived extracellular vesicles (EVs) have gained increasing interest due to their role in disease pathophysiology and parasite biology. Unlike other EV research fields, the isolation of malaria EVs is not standardized, hampering inter-study comparisons. Most malaria EV studies isolate vesicles by the “gold-standard” technique of differential (ultra)centrifugation (DC). Here, we describe in detail an optimized and reproducible protocol for the isolation of malaria-derived EVs by DC. The protocol begins with a description of cultivating high-parasitemia, synchronous P. falciparum cultures that are the source of EV-containing conditioned culture media. The isolation protocol generates two EV subtypes, and we provide details of characterizing these distinct subtypes by analyzing human and parasite proteins by Western blot analysis. We identify some of these proteins as suitable markers for malaria EV subpopulations and subtypes.

Abstract Malaria is a complex parasitic disease, caused by Plasmodium spp. More than a century after the discovery of malaria parasites, this disease continues to pose a global public health problem and the pathogenesis of the severe forms of malaria remains incompletely understood. Extracellular vesicles (EVs), including exosomes and microvesicles, have been increasingly researched in the field of malaria in a bid to fill these knowledge gaps. EVs released from Plasmodium-infected red blood cells and other host cells during malaria infection are now believed to play key roles in disease pathogenesis and are suggested as vital components of the biology of Plasmodium spp. Malaria-derived EVs have been identified as potential disease biomarkers and therapeutic tools. In this review, key findings of malaria EV studies over the last 20 years are summarized and critically analysed. Outstanding areas of research into EV biology are identified. Unexplored EV research foci for the future that will contribute to consolidating the potential for EVs as agents in malaria prevention and control are proposed.

Abstract The Plasmodium falciparum M1 alanyl aminopeptidase and M17 leucyl aminopeptidase, PfM1AAP and PfM17LAP, are potential targets for novel anti-malarial drug development. Inhibitors of these aminopeptidases have been shown to kill malaria parasites in culture and reduce parasite growth in murine models. The two enzymes may function in the terminal stages of haemoglobin digestion, providing free amino acids for protein synthesis by the rapidly growing intra-erythrocytic parasites. Here we have performed a comparative cellular and biochemical characterisation of the two enzymes. Cell fractionation and immunolocalisation studies reveal that both enzymes are associated with the soluble cytosolic fraction of the parasite, with no evidence that they are present within other compartments, such as the digestive vacuole (DV). Enzyme kinetic studies show that the optimal pH of both enzymes is in the neutral range (pH 7.0–8.0), although PfM1AAP also possesses some activity (

Abstract Background Malaria continues to be endemic in the coast and Amazon regions of Ecuador. Clarifying current Plasmodium falciparum resistance in the country will support malaria elimination efforts. In this study, Ecuadorian P. falciparum parasites were analysed to determine their drug resistance genotypes and phenotypes. Methods Molecular analyses were performed to search for mutations in known resistance markers (Pfcrt, Pfdhfr, Pfdhps, Pfmdr1, k13). Pfmdr1 copy number was determined by qPCR. PFMDR1 transporter activity was characterized in live parasites using live cell imaging in combination with the Fluo-4 transport assay. Chloroquine, quinine, lumefantrine, mefloquine, dihydroartemisinin, and artemether sensitivities were measured by in vitro assays. Results The majority of samples from this study presented the CVMNT genotype for Pfcrt (72–26), NEDF SDFD mutations in Pfmdr1 and wild type genotypes for Pfdhfr, Pfdhps and k13. The Ecuadorian P. falciparum strain ESM-2013 showed in vitro resistance to chloroquine, but sensitivity to quinine, lumefantrine, mefloquine, dihydroartemisinin and artemether. In addition, transport of the fluorochrome Fluo-4 from the cytosol into the digestive vacuole (DV) of the ESM-2013 strain was minimally detected in the DV. All analysed samples revealed one copy of Pfmdr1. Conclusion This study indicates that Ecuadorian parasites presented the genotype and phenotype for chloroquine resistance and were found to be sensitive to SP, artemether-lumefantrine, quinine, mefloquine, and dihydroartemisinin. The results suggest that the current malaria treatment employed in the country remains effective. This study clarifies the status of anti-malarial resistance in Ecuador and informs the P. falciparum elimination campaigns in the country.

Abstract Background Chloroquine (CQ) was the drug of choice for decades in the treatment of falciparum malaria until resistance emerged. CQ is suggested to accumulate in the parasite’s digestive vacuole (DV), where it unfolds its anti-malarial properties. Discrepancies of CQ accumulation in CQ-sensitive (CQS) and CQ-resistant (CQR) strains are thought to play a significant role in drug susceptibility. Analysis of CQ transport and intracellular localization using a fluorescently tagged CQ analogue could provide much needed information to distinguish susceptible from resistant parasite strains. The fluorescently tagged CQ analogue LynxTag-CQ™GREEN (CQGREEN) is commercially available and was assessed for its suitability. Methods IC50 values were determined for both CQ and CQGREEN in two CQS and two CQR Plasmodium falciparum strains. Buffer solutions with varying pH were used to determine pH-dependent localization of CQGREEN in infected red blood cells. Before CQS or CQR parasites were exposed to different pH buffers, they were pre-loaded with varying concentrations of CQGREEN for up to 7 h. Intracellular accumulation was analysed using live cell confocal microscopy. CQGREEN uptake rates were determined for the cytosol and DV in the presence and absence of verapamil. Results In CQS strains, twofold higher IC50 values were determined for the CQGREEN analogue compared to CQ. No significant differences in IC50 values were observed in CQR strains. Addition of verapamil reversed drug resistance of CQR strains to both CQ and CQGREEN. Live cell imaging revealed that CQGREEN fluorescence was mainly seen in the cytosol of most parasites, independent of the concentration used. Incubation periods of up to 7 h did not influence intracellular localization of CQGREEN. Nevertheless, CQGREEN uptake rates in CQR strains were reduced by 50% compared to CQS strains. Conclusion Although fluorescence of CQGREEN was mainly seen in the cytosol of parasites, IC50 assays showed comparable efficacy of CQGREEN and CQ in parasite killing of CQS and CQR strains. Reduced uptake rates of CQGREEN in CQR strains compared to CQS strains indicate parasite-specific responses to CQGREEN exposure. The data contains valuable information when CQGREEN is used as an analogue for CQ.

Eradication of malaria, a mosquito-borne parasitic disease that hijacks human red blood cells, is a global priority. Microscopy remains the gold standard hallmark for diagnosis and estimation of parasitemia for malaria, to date. However, this approach is time-consuming and requires much expertise especially in malaria-endemic countries or in areas with low-density malaria infection. Thus, there is a need for accurate malaria diagnosis/parasitemia estimation with standardized, fast, and more reliable methods. To this end, we performed a proof-of-concept study using the automated imaging (NanoZoomer) platform to detect the malarial parasite in infected blood. The approach can be used as a steppingstone for malaria diagnosis and parasitemia estimation. Additionally, we created an algorithm (ParasiteMacro) compatible with free online imaging software (ImageJ) that can be used with low magnification objectives (e.g., 5×, 10×, and 20×) both in the NanoZoomer and routine microscope. The novel approach to estimate malarial parasitemia based on modern technologies compared to manual light microscopy demonstrated 100% sensitivity, 87% specificity, a 100% negative predictive value (NPV) and a 93% positive predictive value (PPV). The manual and automated malaria counts showed a good Pearson correlation for low- (R2 = 0.9377, r = 0.9683 and p < 0.0001) as well as high- parasitemia (R2 = 0.8170, r = 0.9044 and p < 0.0001) with low estimation errors. Our robust strategy that identifies and quantifies malaria can play a pivotal role in disease control strategies.

Malaria is caused by obligate intracellular parasites belonging to the genus Plasmodium. Red blood cells (RBCs) infected with different stages of Plasmodium spp. release extracellular vesicles (EVs). Extensive studies have recently shown that these EVs are involved in key aspects of the parasite’s biology and disease pathogenesis. However, they are yet to be fully characterized. The blood stages of Plasmodium spp., namely the rings, trophozoites and schizonts, are phenotypically distinct, hence, may induce the release of characteristically different EVs from infected RBCs. To gain insights into the biology and biogenesis of malaria EVs, it is important to characterize their biophysical and biochemical properties. By differential centrifugation, we isolated EVs from in vitro cultures of RBCs infected with different stages of Plasmodium falciparum. We performed a preliminary characterization of these EVs and observed that important EV markers were differentially expressed in EVs with different sedimentation properties as well as across EVs released from ring-, trophozoite- or schizont-infected RBCs. Our findings show that RBCs infected with different stages of malaria parasites release EVs with distinct protein expression profiles.

Drug resistance often emerges from mutations in solute transporters. Single amino acid exchanges may alter functionality of transporters with ‘de novo’ ability to transport drugs away from their site of action. The PfMDR1 transporter (or P-glycoprotein 1) is located in the membrane of the digestive vacuole (DV), functions as an ATP-dependent pump, and transports substrates into the DV. In this study, four strains of Plasmodium falciparum, carrying various pfmdr1 gene mutations, were analysed for their transport characteristics of Fluo-4 in isolated DVs of parasites. To obtain quantitative estimates for PfMDR1 DV surface expression, PfMDR1 protein amounts on each strain’s DV membrane were evaluated by quantitative ELISA. Fluo-4, acting as a substrate for PfMDR1, was applied in DV uptake assays (‘reverse Ca2+ imaging’). Viable DVs were isolated from trophozoite stages with preserved PfMDR1 activity. This newly developed assay enabled us to measure the number of Fluo-4 molecules actively transported into isolated DVs per PfMDR1 molecule. The drug-resistant strain Dd2 presented the highest transport rates, followed by K1 and the drug-sensitive strain 3D7, compatible with their copy numbers. With this assay, an evaluation of the probability of resistance formation for newly developed drugs can be implemented in early stages of drug development.