Blood-stage Plasmodium Research Articles

Identification of a divalent metal transporter required for cellular iron metabolism in malaria parasites

Plasmodium falciparum malaria parasites invade and multiply inside red blood cells (RBCs), the most iron-rich compartment in humans. Like all cells, P. falciparum requires nutritional iron to support essential metabolic pathways, but the critical mechanisms of iron acquisition and trafficking during RBC infection have remained obscure. Parasites internalize and liberate massive amounts of heme during large-scale digestion of RBC hemoglobin within an acidic food vacuole (FV) but lack a heme oxygenase to release porphyrin-bound iron. Although most FV heme is sequestered into inert hemozoin crystals, prior studies indicate that trace heme escapes biomineralization and is susceptible to nonenzymatic degradation within the oxidizing FV environment to release labile iron. Parasites retain a homolog of divalent metal transporter 1 (DMT1), a known mammalian iron transporter, but its role in P. falciparum iron acquisition has not been tested. Our phylogenetic studies indicate that P. falciparum DMT1 (PfDMT1) retains conserved molecular features critical for metal transport. We localized this protein to the FV membrane and defined its orientation in an export-competent topology. Conditional knockdown of PfDMT1 expression is lethal to parasites, which display broad cellular defects in iron-dependent functions, including impaired apicoplast biogenesis and mitochondrial polarization. Parasites are selectively rescued from partial PfDMT1 knockdown by supplementation with exogenous iron, but not other metals. These results support a cellular paradigm whereby PfDMT1 is the molecular gatekeeper to essential iron acquisition by blood-stage malaria parasites and suggest that therapeutic targeting of PfDMT1 may be a potent antimalarial strategy.

The Plasmodium falciparum histone methyltransferase SET10 participates in a chromatin modulation network crucial for intraerythrocytic development.

The lifecycle progression of the malaria parasite Plasmodium falciparum requires precise tuning of gene expression including histone methylation. The histone methyltransferase PfSET10 was previously described as an H3K4 methyltransferase involved in var gene regulation, making it a prominent antimalarial target. In this study, we investigated the role of PfSET10 in the blood stages of P. falciparum in more detail, using tagged PfSET10-knockout (KO) and -knockdown (KD) lines. We demonstrate a nuclear localization of PfSET10 with peak protein levels in schizonts. PfSET10 deficiency reduces intraerythrocytic growth but has no effect on gametocyte commitment and maturation. Screening of the PfSET10-KO line for histone methylation variations reveals that lack of PfSET10 renders the parasites unable to mark H3K18me1, while no reduction in the H3K4 methylation status could be observed. Comparative transcriptomic profiling of PfSET10-KO schizonts shows an upregulation of transcripts particularly encoding proteins linked to red blood cell remodeling and antigenic variation, suggesting a repressive function of the histone methylation mark. TurboID coupled with mass spectrometry further highlights an extensive nuclear PfSET10 interaction network with roles in transcriptional regulation and mRNA processing, DNA replication and repair, and chromatin remodeling. The main interactors of PfSET10 include ApiAP2 transcription factors, epigenetic regulators like PfHDAC1, chromatin modulators like PfMORC and PfISWI, mediators of RNA polymerase II, and DNA replication licensing factors. The combined data pinpoint PfSET10 as a histone methyltransferase essential for H3K18 methylation that regulates nucleic acid metabolic processes in the P. falciparum blood stages as part of a comprehensive chromatin modulation network.IMPORTANCEThe fine-tuned regulation of DNA replication and transcription is particularly crucial for the rapidly multiplying blood stages of malaria parasites and proteins involved in these processes represent important drug targets. This study demonstrates that contrary to previous reports the histone methyltransferase PfSET10 of the malaria parasite Plasmodium falciparum promotes the methylation of histone 3 at lysine K18, a histone mark to date not well understood. Deficiency of PfSET10 due to genetic knockout affects genes involved in intraerythrocytic development. Furthermore, in the nuclei of blood-stage parasites, PfSET10 interacts with various protein complexes crucial for DNA replication, remodeling, and repair, as well as for transcriptional regulation and mRNA processing. In summary, this study highlights PfSET10 as a methyltransferase affecting H3K18 methylation with critical functions in chromatin maintenance during the development of P. falciparum in red blood cells.

Progressive heterogeneity of enlarged and irregularly shaped apicoplasts in P. falciparum persister blood stages after drug treatment.

Morphological modifications and shifts in organelle relationships are hallmarks of dormancy in eukaryotic cells. Communications between altered mitochondria and nuclei are associated with metabolic quiescence of cancer cells that can survive chemotherapy. In plants, changes in the pathways between nuclei, mitochondria, and chloroplasts are associated with cold stress and bud dormancy. Plasmodium falciparum parasites, the deadliest agent of malaria in humans, contain a chloroplast-like organelle (apicoplast) derived from an ancient photosynthetic symbiont. Antimalarial treatments can fail because a small fraction of the blood stage parasites enter dormancy and recrudesce after drug exposure. Altered mitochondrial-nuclear interactions in these persisters have been described for P. falciparum, but interactions of the apicoplast remained to be characterized. In the present study, we examined the apicoplasts of persisters obtained after exposure to dihydroartemisinin (a first-line antimalarial drug) followed by sorbitol treatment, or after exposure to sorbitol treatment alone. As previously observed, the mitochondrion of persisters was consistently enlarged and in close association with the nucleus. In contrast, the apicoplast varied from compact and oblate, like those of active ring stage parasites, to enlarged and irregularly shaped. Enlarged apicoplasts became more prevalent later in dormancy, but regular size apicoplasts subsequently predominated in actively replicating recrudescent parasites. All three organelles, nucleus, mitochondrion, and apicoplast, became closer during dormancy. Understanding their relationships in erythrocytic-stage persisters may lead to new strategies to prevent recrudescences and protect the future of malaria chemotherapy.

Identification of a divalent metal transporter required for cellular iron metabolism in malaria parasites.

Plasmodium falciparum malaria parasites invade and multiply inside red blood cells (RBCs), the most iron-rich compartment in humans. Like all cells, P. falciparum requires nutritional iron to support essential metabolic pathways, but the critical mechanisms of iron acquisition and trafficking during RBC infection have remained obscure. Parasites internalize and liberate massive amounts of heme during large-scale digestion of RBC hemoglobin within an acidic food vacuole (FV) but lack a heme oxygenase to release porphyrin-bound iron. Although most FV heme is sequestered into inert hemozoin crystals, prior studies indicate that trace heme escapes biomineralization and is susceptible to non-enzymatic degradation within the oxidizing FV environment to release labile iron. Parasites retain a homolog of divalent metal transporter 1 (DMT1), a known mammalian iron transporter, but its role in P. falciparum iron acquisition has not been tested. Our phylogenetic studies indicate that P. falciparum DMT1 (PfDMT1) retains conserved molecular features critical for metal transport. We localized this protein to the FV membrane and defined its orientation in an export-competent topology. Conditional knockdown of PfDMT1 expression is lethal to parasites, which display broad cellular defects in iron-dependent functions, including impaired apicoplast biogenesis and mitochondrial polarization. Parasites are selectively rescued from partial PfDMT1 knockdown by supplementation with exogenous iron, but not other metals. These results support a cellular paradigm whereby PfDMT1 is the molecular gatekeeper to essential iron acquisition by blood-stage malaria parasites and suggest that therapeutic targeting of PfDMT1 may be a potent antimalarial strategy.

An ApiAp2 Transcription Factor with a Dispensable Role in Plasmodium berghei Life Cycle.

Malaria parasites have a complex life cycle and undergo replication and population expansion within vertebrate hosts and mosquito vectors. These developmental transitions rely on changes in gene expression and chromatin reorganization that result in the activation and silencing of stage-specific genes. The ApiAp2 family of DNA-binding proteins plays an important role in regulating gene expression in malaria parasites. Here, we characterized the ApiAp2 protein in Plasmodium berghei, which we termed Ap2-D. In silico analysis revealed that Ap2-D has three beta-sheets followed by a helix at the C-terminus for DNA binding. Using gene tagging with 3XHA-mCherry, we found that Ap2-D is expressed in Plasmodium blood stages and is present in the parasite cytoplasm and nucleus. Surprisingly, our gene deletion study revealed a completely dispensable role for Ap2-D in the entirety of the P. berghei life cycle. Ap2-D KO parasites were found to grow in the blood successfully and progress through the mosquito midgut and salivary glands. Sporozoites isolated from mosquito salivary glands were infective for hepatocytes and achieved similar patency as WT in mice. We emphasize the importance of genetic validation of antimalarial drug targets before progressing them to drug discovery.

Biosynthesis of gold nanoparticles mediated by medicinal phytometabolites: An effective tool against Plasmodium falciparum and human breast cancer cells

Gold nanoparticles (AuNPs) were synthesized using leaf extracts obtained from the medicinal plants Abutilon theophrasti Medik., Origanum majorana L., Euphorbia hirta L., and Senna auriculata (L.) Roxb. The in vitro antiplasmodial and anticancer activities of AuNPs were assessed against blood-stage malaria parasites and human breast cancer cells, respectively. AuNPs were examined using UV-VIS spectroscopy, HRTEM, DLS, zeta potential, XRD, and FTIR. All the AuNPs exhibited the surface plasmon resonance (SPR) band around 530 nm, which supports the formation of gold particles. The FTIR indicated possible bioactive molecules as constituents of the AuNPs formed. The fluorescence based SYBR Green I assay was utilized to investigate the anti-plasmodial action of AuNPs on the erythrocytic phases of 3D7 (chloroquine drug-sensitive) and INDO (chloroquine drug-resistant) strains of Plasmodium falciparum. The anti-plasmodial IC50 values of the synthesized A. theophrasti AuNPs (N1), O. majorana AuNPs (N2), E. hirta AuNPs (N3), and S. auriculata AuNPs (N4) were 76, 60, 32, and 20 against Pf3D7, and >100, 48, 72, and 36 μg/mL against PfINDO, respectively. N4 showed the highest activity against both P. falciparum strains and also exhibited a high CC50 (480 μg/mL) against human embryonic kidney (HEK293) cells, which leads to a great selectivity index of 24. The in vitro anticancer potential of AuNPs (N1, N2, N3, and N4) was assessed using MCF-7 cells, and the IC50 values were 8.25, 20.25, 20.40, and 24.49 μg/mL, respectively. The present study demonstrated that AuNPs derived from plant extracts may be useful therapeutic agents for treating human breast cancer and malaria.

Ultra-low volume intradermal administration of radiation-attenuated sporozoites with the glycolipid adjuvant 7DW8-5 completely protects mice against malaria

Radiation-attenuated sporozoite (RAS) vaccines can completely prevent blood stage Plasmodium infection by inducing liver-resident memory CD8+ T cells to target parasites in the liver. Such T cells can be induced by ‘Prime-and-trap’ vaccination, which here combines DNA priming against the P. yoelii circumsporozoite protein (CSP) with a subsequent intravenous (IV) dose of liver-homing RAS to “trap” the activated and expanding T cells in the liver. Prime-and-trap confers durable protection in mice, and efforts are underway to translate this vaccine strategy to the clinic. However, it is unclear whether the RAS trapping dose must be strictly administered by the IV route. Here we show that intradermal (ID) RAS administration can be as effective as IV administration if RAS are co-administrated with the glycolipid adjuvant 7DW8-5 in an ultra-low inoculation volume. In mice, the co-administration of RAS and 7DW8-5 in ultra-low ID volumes (2.5 µL) was completely protective and dose sparing compared to standard volumes (10–50 µL) and induced protective levels of CSP-specific CD8+ T cells in the liver. Our finding that adjuvants and ultra-low volumes are required for ID RAS efficacy may explain why prior reports about higher volumes of unadjuvanted ID RAS proved less effective than IV RAS. The ID route may offer significant translational advantages over the IV route and could improve sporozoite vaccine development.

Annickia affinis (Exell) Versteegh & Sosef methanol stem bark extract, potent fractions and isolated Berberine alkaloid target both blood and liver stages of malaria parasites

Ethnopharmacological relevanceHaving identified Annickia affinis as the most potent antiplasmodial plant constituent in a hepta-herbal Agbo-iba (HHA) formula commonly used to manage malaria in Benin city, Nigeria, we have in this study attempted to identify the specialized metabolites responsible for antiplasmodial activity of A. affinis through anti-blood stage malaria parasite activity guided isolation of potent molecules from its stem bark methanol extract. After that, phenotypic effects, including stage-specific kill kinetics, were investigated. Further, the crude extract, its potent fractions, and specialized metabolites were also tested against the liver-stage malaria parasite. Materials and methodsA. affinis was subjected to molecular PCR-based analysis to confirm its identity. Thereafter, extraction of its stem bark with methanol was carried out. Alkaloid enriched fractions from this stem bark extract were obtained using the acid-base-solvent extraction method. These alkaloid-enriched fractions were subjected to various chromatographic techniques that led to the isolation of two protoberberine alkaloids identified as berberine and palmatine based on NMR and mass spectrometry analysis. The efficacy of crude extract, fractions and purified alkaloids was tested against the malaria parasite's blood and liver stages, respectively. Results and DiscussionAnnickia affinis methanol extract, fractions, and the isolated protoberberine alkaloids showed excellent antiplasmodial activity with good selectivity against blood-stage malaria parasite. Thus, their IC50 against various strains of the parasite ranged from 0.95 to 18.65 μg/ml, while CC50 against Human embryonic kidney (HEK) and the human hepatoma (HUH-7) cell lines ranged between 10 and > 100 μg/ml. Interestingly, the crude extract and the alkaloid enriched fractions showed promising activity against the liver-stage malaria parasite. Between berberine and palmatine isolated from the potent fractions, only the former showed ∼100% and 90% inhibitions of liver stage parasite at 5 μg/ml and 1 μg/ml, respectively, while the latter showed no inhibition even at 20 μg/ml. ConclusionThis study reports that the ethnomedicinal use of HHA to manage malaria can be attributed to the presence of promising antiplasmodial protoberberine alkaloids together with synergistic effects via either enhancement of bioavailability or improved pharmacokinetics by other phytoconstituent(s) coming from other HHA constituent plants. The protoberberine alkaloids isolated have been identified as fast-acting antiplasmodial agents, with activity against all erythrocytic stages of the malaria parasite. Further, A. affinis methanol stembark extract and the protoberberine alkaloid berberine isolated from it also displayed excellent activity (>90% inhibition at 1 μg/ml) against the liver-stage malaria parasite. A. affinis and HHA can thus be useful as both liver-stage prophylactic and blood-stage curative agents.

Essential function of alveolin PfIMC1g in the Plasmodium falciparum asexual blood stage.

Infection by the Plasmodium falciparum parasite is responsible for the most severe form of human malaria. The asexual blood stage of the parasite, which occurs inside human red blood cells, is responsible for the symptoms of malaria and is the target of most antimalarial drugs. Plasmodium spp. rely on their highly divergent cytoskeletal structures to scaffold their cell division, sustain the mechanical stress of invasion, and survive in both the human bloodstream and the mosquito. We investigate the function of a class of divergent intermediate filament-like proteins called alveolins in the clinically important blood stage. The functional role of individual alveolins in Plasmodium remains poorly understood due to pleiotropic effects of gene knockouts and redundancy among alveolins. We evaluate the localization and essentiality of the four asexual-stage alveolins and find that PfIMC1g and PfIMC1c are essential. Furthermore, we demonstrate that PfIMC1g is critical for survival of the parasite post-invasion.

Facile Access to Structurally Diverse Antimalarial Indoles Using a One-Pot A3 Coupling and Domino Cyclization Approach.

A multistep and diversity-oriented synthetic route aiming at the A3 coupling/domino cyclization of o-ethynyl anilines, aldehydes and s-amines is described. The preparation of the corresponding precursors included a series of transformations, such as haloperoxidation and Sonogashira cross-coupling reactions, amine protection, desilylation and amine reduction. Some products of the multicomponent reaction underwent further detosylation and Suzuki coupling. The resulting library of structurally diverse compounds was evaluated against blood and liver stage malaria parasites, which revealed a promising lead with sub-micromolar activity against intra-erythrocytic forms of Plasmodium falciparum. The results from this hit-to-lead optimization are hereby reported for the first time.

A malaria parasite phospholipase facilitates efficient asexual blood stage egress.

Malaria parasite release (egress) from host red blood cells involves parasite-mediated membrane poration and rupture, thought to involve membrane-lytic effector molecules such as perforin-like proteins and/or phospholipases. With the aim of identifying these effectors, we disrupted the expression of two Plasmodium falciparum perforin-like proteins simultaneously and showed that they have no essential roles during blood stage egress. Proteomic profiling of parasite proteins discharged into the parasitophorous vacuole (PV) just prior to egress detected the presence in the PV of a lecithin:cholesterol acyltransferase (LCAT; PF3D7_0629300). Conditional ablation of LCAT resulted in abnormal egress and a reduced replication rate. Lipidomic profiles of LCAT-null parasites showed drastic changes in several phosphatidylserine and acylphosphatidylglycerol species during egress. We thus show that, in addition to its previously demonstrated role in liver stage merozoite egress, LCAT is required to facilitate efficient egress in asexual blood stage malaria parasites.

Activity of Ivermectin and Its Metabolites against Asexual Blood Stage Plasmodium falciparum and Its Interactions with Antimalarial Drugs.

Ivermectin is an endectocide used widely to treat a variety of internal and external parasites. Field trials of ivermectin mass drug administration for malaria transmission control have demonstrated a reduction of Anopheles mosquito survival and human malaria incidence. Ivermectin will mostly be deployed together with artemisinin-based combination therapies (ACT), the first-line treatment of falciparum malaria. It has not been well established if ivermectin has activity against asexual stage Plasmodium falciparum or if it interacts with the parasiticidal activity of other antimalarial drugs. This study evaluated antimalarial activity of ivermectin and its metabolites in artemisinin-sensitive and artemisinin-resistant P. falciparum isolates and assessed in vitro drug-drug interaction with artemisinins and its partner drugs. The concentration of ivermectin causing half of the maximum inhibitory activity (IC50) on parasite survival was 0.81 μM with no significant difference between artemisinin-sensitive and artemisinin-resistant isolates (P = 0.574). The ivermectin metabolites were 2-fold to 4-fold less active than the ivermectin parent compound (P < 0.001). Potential pharmacodynamic drug-drug interactions of ivermectin with artemisinins, ACT-partner drugs, and atovaquone were studied in vitro using mixture assays providing isobolograms and derived fractional inhibitory concentrations. There were no synergistic or antagonistic pharmacodynamic interactions when combining ivermectin and antimalarial drugs. In conclusion, ivermectin does not have clinically relevant activity against the asexual blood stages of P. falciparum. It also does not affect the in vitro antimalarial activity of artemisinins or ACT-partner drugs against asexual blood stages of P. falciparum.

Mosquito bites and stage-specific antibody responses against Plasmodium falciparum in southern Ghana

BackgroundThe human host elicits specific immune responses after exposure to various life stages of the malaria parasite as well as components of mosquito saliva injected into the host during a mosquito bite. This study describes differences in IgG responses against antigens derived from the sporozoite (PfCSP), asexual stage parasite (PfEBA175) and the gametocyte (Pfs230), in addition to an Anopheles gambiae salivary gland antigen (gSG6-P1), in two communities in Ghana with similar blood stage malaria parasite prevalence.MethodsThis study used archived plasma samples collected from an earlier cross-sectional study that enrolled volunteers aged from 6 months to 70 years from Simiw, peri-urban community (N = 347) and Obom, rural community (N = 291). An archived thick and thin blood smear for microscopy was used for the estimation of Plasmodium parasite density and species and DNA extraction from blood spots and P. falciparum confirmation was performed using PCR. This study used the stored plasma samples to determine IgG antibody levels to P. falciparum and Anopheles salivary antigens using indirect ELISA.ResultsIndividuals from Simiw had significantly higher levels of IgG against mosquito gSG6-P1 [median (95%CI)] [2.590 (2.452–2.783) ng/mL] compared to those from Obom [2.119 (1.957–2.345) ng/mL], p < 0.0001. Both IgG responses against Pfs230proC (p = 0.0006), and PfCSP (p = 0.002) were significantly lower in volunteers from Simiw compared to the participants from Obom. The seroprevalence of PfEBA-175.5R (p = 0.8613), gSG6-P1 (p = 0.0704), PfCSP (p = 0.7798) IgG were all similar in Obom and Simiw. However, Pfs230 seroprevalence was significantly higher at Obom compared to Simiw (p = 0.0006). Spearman correlation analysis showed no significant association between IgG responses against gSG6-P1, PfCSP, Pfs230proC and PfEBA-175.5R and parasite density at both Obom and Simiw (p > 0.05).ConclusionIn conclusion, the study showed that participants from Simiw had higher concentrations of circulating gSG6-P1 IgG antibodies but lower concentrations of P. falciparum antibodies, PfCSP IgG and Pfs230proC IgG compared to participants from Obom.

The claudin-like apicomplexan microneme protein is required for gliding motility and infectivity of Plasmodium sporozoites.

Invasion of host cells by apicomplexan parasites such as Toxoplasma and Plasmodium spp requires the sequential secretion of the parasite apical organelles, the micronemes and the rhoptries. The claudin-like apicomplexan microneme protein (CLAMP) is a conserved protein that plays an essential role during invasion by Toxoplasma gondii tachyzoites and in Plasmodium falciparum asexual blood stages. CLAMP is also expressed in Plasmodium sporozoites, the mosquito-transmitted forms of the malaria parasite, but its role in this stage is still unknown. CLAMP is essential for Plasmodium blood stage growth and is refractory to conventional gene deletion. To circumvent this obstacle and study the function of CLAMP in sporozoites, we used a conditional genome editing strategy based on the dimerisable Cre recombinase in the rodent malaria model parasite P. berghei. We successfully deleted clamp gene in P. berghei transmission stages and analyzed the functional consequences on sporozoite infectivity. In mosquitoes, sporozoite development and egress from oocysts was not affected in conditional mutants. However, invasion of the mosquito salivary glands was dramatically reduced upon deletion of clamp gene. In addition, CLAMP-deficient sporozoites were impaired in cell traversal and productive invasion of mammalian hepatocytes. This severe phenotype was associated with major defects in gliding motility and with reduced shedding of the sporozoite adhesin TRAP. Expansion microscopy revealed partial colocalization of CLAMP and TRAP in a subset of micronemes, and a distinct accumulation of CLAMP at the apical tip of sporozoites. Collectively, these results demonstrate that CLAMP is essential across invasive stages of the malaria parasite, and support a role of the protein upstream of host cell invasion, possibly by regulating the secretion or function of adhesins in Plasmodium sporozoites.

Metabolic responses in blood-stage malaria parasites associated with increased and decreased sensitivity to PfATP4 inhibitors

BackgroundSpiroindolone and pyrazoleamide antimalarial compounds target Plasmodium falciparum P-type ATPase (PfATP4) and induce disruption of intracellular Na+ homeostasis. Recently, a PfATP4 mutation was discovered that confers resistance to a pyrazoleamide while increasing sensitivity to a spiroindolone. Transcriptomic and metabolic adaptations that underlie this seemingly contradictory response of P. falciparum to sublethal concentrations of each compound were examined to understand the different cellular accommodation to PfATP4 disruptions.MethodsA genetically engineered P. falciparum Dd2 strain (Dd2A211V) carrying an Ala211Val (A211V) mutation in PfATP4 was used to identify metabolic adaptations associated with the mutation that results in decreased sensitivity to PA21A092 (a pyrazoleamide) and increased sensitivity to KAE609 (a spiroindolone). First, sublethal doses of PA21A092 and KAE609 causing substantial reduction (30–70%) in Dd2A211V parasite replication were identified. Then, at this sublethal dose of PA21A092 (or KAE609), metabolomic and transcriptomic data were collected during the first intraerythrocytic developmental cycle. Finally, the time-resolved data were integrated with a whole-genome metabolic network model of P. falciparum to characterize antimalarial-induced physiological adaptations.ResultsSublethal treatment with PA21A092 caused significant (p < 0.001) alterations in the abundances of 91 Plasmodium gene transcripts, whereas only 21 transcripts were significantly altered due to sublethal treatment with KAE609. In the metabolomic data, a substantial alteration (≥ fourfold) in the abundances of carbohydrate metabolites in the presence of either compound was found. The estimated rates of macromolecule syntheses between the two antimalarial-treated conditions were also comparable, except for the rate of lipid synthesis. A closer examination of parasite metabolism in the presence of either compound indicated statistically significant differences in enzymatic activities associated with synthesis of phosphatidylcholine, phosphatidylserine, and phosphatidylinositol.ConclusionThe results of this study suggest that malaria parasites activate protein kinases via phospholipid-dependent signalling in response to the ionic perturbation induced by the Na+ homeostasis disruptor PA21A092. Therefore, targeted disruption of phospholipid signalling in PA21A092-resistant parasites could be a means to block the emergence of resistance to PA21A092.

In vitro model of brain endothelial cell barrier reveals alterations induced by Plasmodium blood stage factors

Cerebral malaria (CM) is a severe neurological condition caused by Plasmodium falciparum. Disruption of the brain-blood barrier (BBB) is a key pathological event leading to brain edema and vascular leakage in both humans and in the mouse model of CM. Interactions of brain endothelial cells with infected red blood cells (iRBCs) and with circulating inflammatory mediators and immune cells contribute to BBB dysfunction in CM. Adjunctive therapies for CM aim at preserving the BBB to prevent neurologic deficits. Experimental animal and cellular models are essential to develop new therapeutic strategies. However, in mice, the disease develops rapidly, which offers a very narrow time window for testing the therapeutic potential of drugs acting in the BBB. Here, we establish a brain endothelial cell barrier whose disturbance can be monitored by several parameters. Using this system, we found that incubation with iRBCs and with extracellular particles (EPs) released by iRBCs changes endothelial cell morphology, decreases the tight junction protein zonula occludens-1 (ZO-1), increases the gene expression of the intercellular adhesion molecule 1 (ICAM-1), and induces a significant reduction in transendothelial electrical resistance (TEER) with increased permeability. We propose this in vitro experimental setup as a straightforward tool to investigate molecular interactions and pathways causing endothelial barrier dysfunction and to test compounds that may target BBB and be effective against CM. A pre-selection of the effective compounds that strengthen the resistance of the brain endothelial cell barrier to Plasmodium-induced blood factors in vitro may increase the likelihood of their efficacy in preclinical disease mouse models of CM and in subsequent clinical trials with patients.

Plasmodium falciparum Sexual Commitment Rate Variation among Clinical Isolates and Diverse Laboratory-Adapted Lines.

Asexual blood-stage malaria parasites must produce sexual progeny to infect mosquitoes. It is important to understand the scope and causes of intraspecific variation in sexual commitment rates, particularly for the major human parasite P. falciparum. First, two alternative assay methods of measuring sexual commitment were compared to test a genetically modified P. falciparum line with elevated commitment rates inducible by overexpression of GDV1. The methods yielded correlated measurements with higher sensitivity and precision being achieved by one employing detection of the early gametocyte differentiation marker Pfs16. Thus, this was used to survey a diverse range of parasite lines and test each in multiple biological replicate assays in a serum-free medium supplemented with Albumax. There were differences among six recent clinical isolates from Ghana in their mean rates of sexual commitment per cycle, ranging from 3.3% to 12.2%. Among 13 diverse long-term laboratory-adapted lines, mean sexual commitment rates for most ranged from 4.7% to 13.4%, a few had lower rates with means from 0.3 to 1.6%, and one with a nonfunctional ap2-g gene always showed zero commitment. Among a subset of lines tested for the effects of exogenous choline to suppress commitment, there were significant differences. As expected, there was no effect in a line that had lost the gdv1 gene and that had generally low commitment, whereas the others showed quantitatively variable but significant responses to choline, suggesting potential trait variation. The results indicated the value of performing multiple replicate assays for understanding the variation of this key reproductive trait that likely affects transmission. IMPORTANCE Only sexual-stage malaria parasites are transmitted from human blood to mosquitoes. Thus, it is vital to understand variations in sexual commitment rates because these may be modifiable or susceptible to blocking. Two different methods of commitment rate measurement were first compared, demonstrating higher sensitivity and precision by the detection of an early differentiation marker, which was subsequently used to survey diverse lines. Clinical isolates from Ghana showed significant variation in mean per-cycle commitment rates and variation among biological replicates. Laboratory-adapted lines of diverse origins had a wider range with most being within the range observed for the clinical isolates, while a minority consistently had lower or zero rates. There was quantitative variation in the effects when adding choline to suppress commitment, indicating differing responsiveness of parasites to this environmental modification. Performing multiple assay replicates and comparisons of diverse isolates was important to understand this trait and its potential effects on transmission.

Generation of red blood cells from stem cells: Achievements, opportunities and perspectives for malaria research.

Parasites of the genus Plasmodium that cause malaria survive within humans by invasion of, and proliferation within, the most abundant cell type in the body, the red blood cell. As obligate, intracellular parasites, interactions between parasite and host red blood cell components are crucial to multiple aspects of the blood stage malaria parasite lifecycle. The requirement for, and involvement of, an array of red blood cell proteins in parasite invasion and intracellular development is well established. Nevertheless, detailed mechanistic understanding of host cell protein contributions to these processes are hampered by the genetic intractability of the anucleate red blood cell. The advent of stem cell technology and more specifically development of methods that recapitulate in vitro the process of red blood cell development known as erythropoiesis has enabled the generation of erythroid cell stages previously inaccessible in large numbers for malaria studies. What is more, the capacity for genetic manipulation of nucleated erythroid precursors that can be differentiated to generate modified red blood cells has opened new horizons for malaria research. This review summarises current methodologies that harness in vitro erythroid differentiation of stem cells for generation of cells that are susceptible to malaria parasite invasion; discusses existing and emerging approaches to generate novel red blood cell phenotypes and explores the exciting potential of in vitro derived red blood cells for improved understanding the broad role of host red blood cell proteins in malaria pathogenesis.

‘Erythritol’, a safe natural sweetener exhibits multi-stage anti-malarial activity by permeating into Plasmodium falciparum through aquaglyceroporin channel

The increased resistance of human malaria parasite Plasmodium falciparum (Pf) to currently used drugs necessities the development of novel anti-malarials. Here, we examine the potential of erythritol, a sugar substitute for therapeutic intervention. Erythritol is a permeant of Plasmodium falciparum aquaglyceroporin (PfAQP) which is a multifunctional channel responsible for maintaining hydro-homeostasis. We show that erythritol effectively inhibited growth and progression of asexual blood stage malaria parasite, and effect invasion and egress processes. It also inhibited the liver stage (sporozoites) and transmission stage parasite (gametocytes) development. Interestingly, erythritol inhibited in vivo growth of malaria parasite in mouse experimental model. It was more effective in inhibiting parasite growth both in vivo and in vitro when tested together with a known anti-malarial ‘artesunate’. Additionally, erythritol showed cytokine-modulating effect which suggests its direct effect on the host immune system. Ammonia detection assay demonstrated that erythritol uptake effects the amount of ammonia release across the parasite. Our functional complementation assays suggest that PfAQP expression in yeast mutant restores its growth in hyperosmotic conditions but showed reduced growth in the presence of erythritol. Osmotic lysis assay suggests that erythritol creates osmotic stress for killing the parasite. Overall, our data bestow erythritol as a promising lead compound with an attractive antimalarial profile and could possibly be combined with known drugs without losing its efficacy. We propose the use of erythritol based sweet candies for protection against malaria specially in children living in the endemic area.

Mannose inhibits Plasmodium parasite growth and cerebral malaria developmentvia regulation of host immune responses.

D-mannose can be transported into a variety of cells via glucose transporter (GLUT), and supraphysiological levels of D-mannose impairs tumor growth and modulates immune cell function through mechanisms such as interference with glycolysis and induction of oxidative stress. Blood-stage Plasmodium mainly depends on glycolysis for energy supply and pathological immune response plays a vital role in cerebral malaria. However, it is not clear whether mannose affects malaria blood-stage infection. Here, we fed D-mannose to Plasmodium berghei-infected mice and found weight loss and reduced parasitemia without apparent side effects. Compromised parasitemia in C57BL/6 mice was accompanied by an increase in splenic macrophages compared to an untreated group. When mannose was applied to a rodent experimental cerebral malaria (ECM) model, the incidence of ECM decreased. Expression of activation marker CD69 on T cells in peripheral blood and the brain were reduced, and cerebral migration of activated T cells was prevented by decreased expression of CXCR3. These findings suggest that mannose inhibits Plasmodium infection by regulating multiple host immune responses and could serve as a potential strategy for facilitating malaria treatment.