Inner membrane complex proteomics reveals a palmitoylation regulation critical for intraerythrocytic development of malaria parasite.

Malaria is caused by infection of the erythrocytes by the parasites Plasmodium. Inside the erythrocytes, the parasites multiply via schizogony, an unconventional cell division mode. The inner membrane complex (IMC), an organelle located beneath the parasite plasma membrane, serving as the platform for protein anchorage, is essential for schizogony. So far, the complete repertoire of IMC proteins and their localization determinants remain unclear. Here we used biotin ligase (TurboID)-based proximity labeling to compile the proteome of the schizont IMC of the rodent malaria parasite Plasmodium yoelii. In total, 300 TurboID-interacting proteins were identified. 18 of 21 selected candidates were confirmed to localize in the IMC, indicating good reliability. In light of the existing palmitome of Plasmodium falciparum, 83 proteins of the P. yoelii IMC proteome are potentially palmitoylated. We further identified DHHC2 as the major resident palmitoyl-acyl-transferase of the IMC. Depletion of DHHC2 led to defective schizont segmentation and growth arrest both in vitro and in vivo. DHHC2 was found to palmitoylate two critical IMC proteins CDPK1 and GAP45 for their IMC localization. In summary, this study reports an inventory of new IMC proteins and demonstrates a central role of DHHC2 in governing the IMC localization of proteins during the schizont development.

Screening the Medicines for Malaria Venture Pathogen Box for invasion and egress inhibitors of the blood stage of Plasmodium falciparum reveals several inhibitory compounds.

With emerging resistance to frontline treatments, it is vital that new drugs are identified to target Plasmodium falciparum. One of the most critical processes during parasites asexual lifecycle is the invasion and subsequent egress of red blood cells (RBCs). Many unique parasite ligands, receptors and enzymes are employed during egress and invasion that are essential for parasite proliferation and survival, therefore making these processes druggable targets. To identify potential inhibitors of egress and invasion, we screened the Medicines for Malaria Venture Pathogen Box, a 400 compound library against neglected tropical diseases, including 125 with antimalarial activity. For this screen, we utilised transgenic parasites expressing a bioluminescent reporter, nanoluciferase (Nluc), to measure inhibition of parasite egress and invasion in the presence of the Pathogen Box compounds. At a concentration of 2 µM, we found 15 compounds that inhibited parasite egress by >40% and 24 invasion-specific compounds that inhibited invasion by >90%. We further characterised 11 of these inhibitors through cell-based assays and live cell microscopy, and found two compounds that inhibited merozoite maturation in schizonts, one compound that inhibited merozoite egress, one compound that directly inhibited parasite invasion and one compound that slowed down invasion and arrested ring formation. The remaining compounds were general growth inhibitors that acted during the egress and invasion phase of the cell cycle. We found the sulfonylpiperazine, MMV020291, to be the most invasion-specific inhibitor, blocking successful merozoite internalisation within human RBCs and having no substantial effect on other stages of the cell cycle. This has significant implications for the possible development of an invasion-specific inhibitor as an antimalarial in a combination based therapy, in addition to being a useful tool for studying the biology of the invading parasite.

Whole-Cell Phenotypic Screening of Medicines for Malaria Venture Pathogen Box Identifies Specific Inhibitors of Plasmodium falciparum Late-Stage Development and Egress.

We report a systematic, cellular phenotype-based antimalarial screening of the Medicines for Malaria Venture Pathogen Box collection, which facilitated the identification of specific blockers of late-stage intraerythrocytic development of Plasmodium falciparum First, from standard growth inhibition assays, we identified 173 molecules with antimalarial activity (50% effective concentration [EC50] ≤ 10 µM), which included 62 additional molecules over previously known antimalarial candidates from the Pathogen Box. We identified 90 molecules with EC50 of ≤1 µM, which had significant effect on the ring-trophozoite transition, while 9 molecules inhibited the trophozoite-schizont transition and 21 molecules inhibited the schizont-ring transition (with ≥50% parasites failing to proceed to the next stage) at 1 µM. We therefore rescreened all 173 molecules and validated hits in microscopy to prioritize 12 hits as selective blockers of the schizont-ring transition. Seven of these molecules inhibited the calcium ionophore-induced egress of Toxoplasma gondii, a related apicomplexan parasite, suggesting that the inhibitors may be acting via a conserved mechanism which could be further exploited for target identification studies. We demonstrate that two molecules, MMV020670 and MMV026356, identified as schizont inhibitors in our screens, induce the fragmentation of DNA in merozoites, thereby impairing their ability to egress and invade. Further mechanistic studies would facilitate the therapeutic exploitation of these molecules as broadly active inhibitors targeting late-stage development and egress of apicomplexan parasites relevant to human health.

The impact of early life exposure to Plasmodium falciparum on the development of naturally acquired immunity to malaria in young Malawian children.

BACKGROUND: Antibodies targeting malaria blood-stage antigens are important targets of naturally acquired immunity, and may act as valuable biomarkers of malaria exposure. METHODS: Six-hundred and one young Malawian children from a randomized trial of prenatal nutrient supplementation with iron and folic acid or pre- and postnatal multiple micronutrients or lipid-based nutrient supplements were followed up weekly at home and febrile episodes were investigated for malaria from birth to 18 months of age. Antibodies were measured for 601 children against merozoite surface proteins (MSP1 19kD, MSP2), erythrocyte binding antigen 175 (EBA175), reticulocyte binding protein homologue 2 (Rh2A9), schizont extract and variant surface antigens expressed by Plasmodium falciparum-infected erythrocytes (IE) at 18 months of age. The antibody measurement data was related to concurrent malaria infection and to documented episodes of clinical malaria. RESULTS: At 18 months of age, antibodies were significantly higher among parasitaemic than aparasitaemic children. Antibody levels against MSP1 19kD, MSP2, schizont extract, and IE variant surface antigens were significantly higher in children who had documented episodes of malaria than in children who did not. Antibody levels did not differ between children with single or multiple malaria episodes before 18 months, nor between children who had malaria before 6 months of age or between 6 and 18 months. CONCLUSIONS: Antibodies to merozoite and IE surface antigens increased following infection in early childhood, but neither age at first infection nor number of malaria episodes substantially affected antibody acquisition. These findings have implications for malaria surveillance during early childhood in the context of elimination. Trials registration Clinical Trials Registration: NCT01239693 (Date of registration: 11-10-2010). URL: http://www.ilins.org.

Spirocyclic chromanes exhibit antiplasmodial activities and inhibit all intraerythrocytic life cycle stages.

We screened a collection of synthetic compounds consisting of natural-product-like substructural motifs to identify a spirocyclic chromane as a novel antiplasmodial pharmacophore using an unbiased cell-based assay. The most active spirocyclic compound UCF 201 exhibits a 50% effective concentration (EC50) of 350 nM against the chloroquine-resistant Dd2 strain and a selectivity over 50 using human liver HepG2 cells. Our analyses of physicochemical properties of UCF 201 showed that it is in compliance with Lipinski's parameters and has an acceptable physicochemical profile. We have performed a limited structure-activity-relationship study with commercially available chromanes preserving the spirocyclic motif. Our evaluation of stage specificities of UCF 201 indicated that the compound is early-acting in blocking parasite development at ring, trophozoite and schizont stages of development as well as merozoite invasion. SPC is an attractive lead candidate scaffold because of its ability to act on all stages of parasite's aexual life cycle unlike current antimalarials.

The impact of lipid-based nutrient supplementation on anti-malarial antibodies in pregnant women in a randomized controlled trial.

BACKGROUND: Malaria and undernutrition frequently coexist, especially in pregnant women and young children. Nutrient supplementation of these vulnerable groups might reduce their susceptibility to malaria by improving immunity. METHODS: Antibody immunity to antigens expressed by a placental-binding parasite isolate, a non-placental binding parasite isolate, merozoites and schizonts at enrolment (before 20 gestation weeks) and at 36 gestation weeks were measured in 1,009 Malawian pregnant women receiving a daily lipid-based nutrient supplement, multiple micronutrients or iron and folic acid, who were participants in a randomized clinical trial assessing the effects of nutrient supplementation on pregnancy outcomes and child development (registration ID: NCT01239693). RESULTS: Antibodies to placental-binding isolates significantly increased while antibodies to most merozoite antigens declined over pregnancy. Overall, after adjustment for covariates, the type of supplementation did not influence antibody levels at 36 gestation weeks or the rate of change in antibody levels from enrolment to 36 weeks. A negative association between maternal body mass index and opsonizing antibodies to placental-binding antigens (coefficient (95% CI) -1.04 (-1.84, -0.24), was observed. Similarly, women with higher socioeconomic status had significantly lower IgG and opsonizing antibodies to placental-binding antigens. Neither of these associations was significantly influenced by the supplementation type. CONCLUSIONS: In the current cohort nutrient supplementation did not affect anti-malarial antibody responses, but poor and undernourished mothers should be a priority group in future trials.

Azadirachta indica extract-artesunic acid combination produces an increased cure rate of Plasmodium berghei-infected mice.

CONTEXT: Available artemisinin-combination therapies (ACTs) are expensive. Various traditional herbal remedies for malaria involve plants, proven scientifically to have antiplasmodial effects, e.g., Azadirachta indica A. Juss. (Meliaceae). OBJECTIVE: Combination of an artemisinin-based compound and a medicinal herb extract will provide an indigenous alternative/herb-based ACT. MATERIALS AND METHODS: The in vivo schizontocidal activity of the crude aqueous extract of 100, 500, and 1000 mg/kg of A. indica fresh leaves (NCE) and 6, 15, and 20 mg/kg of artesunic acid were determined, alone and in combination, while keeping the dose of artesunic acid constant at 15 mg/kg, using the Peter's 4-day suppressive test and Swiss albino mice. The ED50 was calculated from the dose-response relationships. Percentage survival and cure were also determined. RESULTS: The average yield of two extractions of NCE was 8.33 ± 1.67%. Combination of 1000 mg/kg of NCE and 15 mg/kg of artesunic acid, produced a significant reduction of parasitemia (96.87%), compared to 20 mg/kg of artesunic acid alone (68.14%). The combination had an ED50 of 0.58 mg/kg while that of artesunic acid alone was 8.814 mg/kg. The combinations of NCE with artesunic acid produced a cure, although the artesunic acid did not produce a cure in 30 d. DISCUSSION: NCE increased the activity of artesunic acid in terms of reduction in parasitemia, and increased survival time and cure rate. CONCLUSION: The combination of an artemisinin and aqueous extract of neem leaf is possible, providing a potentiated reduction of parasitemia, and increased cure rate.

Broad-spectrum antimalarial activity of peptido sulfonyl fluorides, a new class of proteasome inhibitors.

Despite declining numbers of cases and deaths, malaria remains a major public health problem in many parts of the world. Today, case management relies heavily on a single class of antimalarial compounds: artemisinins. Hence, development of resistance against artemisinins may destroy current malaria control strategies. Beyond malaria control are elimination and eradication programs that will require drugs with good activity against acute infection but also with preventive and transmission-blocking properties. Consequently, new antimalarials are needed not only to ensure malaria control but also for elimination and eradication efforts. In this study, we introduce peptido sulfonyl fluorides (PSF) as a new class of compounds with antiplasmodial activity. We show that PSF target the plasmodial proteasome and act on all asexual stages of the intraerythrocytic cycle and on gametocytes. PSF showed activities at concentrations as low as 20 nM against multidrug-resistant and chloroquine-sensitive Plasmodium falciparum laboratory strains and clinical isolates from Gabon. Structural requirements for activity were identified, and cytotoxicity in human HeLa or HEK 293 cells was low. The lead PSF PW28 suppressed growth of Plasmodium berghei in vivo but showed signs of toxicity in mice. Considering their modular structure and broad spectrum of activity against different stages of the plasmodial life cycle, proteasome inhibitors based on PSF have a great potential for further development as preclinical candidate compounds with improved species-specific activity and less toxicity.

Insights into the preclinical treatment of blood-stage malaria by the antibiotic borrelidin.

BACKGROUND AND PURPOSE: Blood-stage Plasmodium parasites cause morbidity and mortality from malaria. Parasite resistance to drugs makes development of new chemotherapies an urgency. Aminoacyl-tRNA synthetases have been validated as antimalarial drug targets. We explored long-term effects of borrelidin and mupirocin in lethal P. yoelii murine malaria. EXPERIMENTAL APPROACH: Long-term (up to 340 days) immunological responses to borrelidin or mupirocin were measured after an initial 4 day suppressive test. Prophylaxis and cure were evaluated and the inhibitory effect on the parasites analysed. KEY RESULTS: Borrelidin protected against lethal malaria at 0.25 mg·kg⁻¹·day⁻¹. Antimalarial activity of borrelidin correlated with accumulation of trophozoites in peripheral blood. All infected mice treated with borrelidin survived and subsequently developed immunity protecting them from re-infection on further challenges, 75 and 340 days after the initial infection. This long-term immunity in borrelidin-treated mice resulted in negligible parasitaemia after re-infections and marked increases in total serum levels of antiparasite IgGs with augmented avidity. Long-term memory IgGs mainly reacted against high and low molecular weight parasite antigens. Immunofluorescence microscopy showed that circulating IgGs bound predominantly to late intracellular stage parasites, mainly schizonts. CONCLUSIONS AND IMPLICATIONS: Low borrelidin doses protected mice from lethal malaria infections and induced protective immune responses after treatment. Development of combination therapies with borrelidin and selective modifications of the borrelidin molecule to specifically inhibit plasmodial threonyl tRNA synthetase should improve therapeutic strategies for malaria.

Parasite maturation and host serum iron influence the labile iron pool of erythrocyte stage Plasmodium falciparum.

Iron is a critical and tightly regulated nutrient for both the malaria parasite and its human host. The importance of the relationship between host iron and the parasite has been underscored recently by studies showing that host iron supplementation may increase the risk of falciparum malaria. It is unclear what host iron sources the parasite is able to access. We developed a flow cytometry-based method for measuring the labile iron pool (LIP) of parasitized erythrocytes using the nucleic acid dye STYO 61 and the iron sensitive dye, calcein acetoxymethyl ester (CA-AM). This new approach enabled us to measure the LIP of P. falciparum through the course of its erythrocytic life cycle and in response to the addition of host serum iron sources. We found that the LIP increases as the malaria parasite develops from early ring to late schizont stage, and that the addition of either transferrin or ferric citrate to culture media increases the LIP of trophozoites. Our method for detecting the LIP within malaria parasitized RBCs provides evidence that the parasite is able to access serum iron sources as part of the host vs. parasite arms race for iron.

Dehydroepiandrosterone effect on Plasmodium falciparum and its interaction with antimalarial drugs.

Dehydroepiandrosterone (DHEA) inhibits glucose 6-phosphate dehydrogenase (G6PDH) of different species and may potentially decrease intracellular glutathione. Therefore, it can have and enhance anti-parasitic action against Plasmodium spp. We evaluated the antiplasmodial activity and the interaction of DHEA with several antimalarial drugs. The inhibitory effect of DHEA on erythrocytic and G6PDH activity and changes in the content of total and reduced gluthatione Plasmodium falciparum content were also evaluated. DHEA showed antiplasmodial activity in vitro, but the potency was low (IC(50) 118.5 µM). DHEA inhibits G6PDH from healthy erythrocyte and decreases GSH content in both erythrocytes and P. falciparum. DHEA did not synergize or antagonize the antiplasmodial effect of several antimalarial drugs. The most important actions of DHEA were the inhibition of G6PDH activity, and the decrease in both P. falciparum and erythrocyte GSH. Since most of the GSH in Plasmodium spp. infected erythrocytes comes from the parasite itself, it is possible that DHEA analogs could act with higher selectivity, better potency, and might interact synergistically with antimalarials drugs.

Impairment of the Plasmodium falciparum erythrocytic cycle induced by angiotensin peptides.

Plasmodium falciparum causes the most serious complications of malaria and is a public health problem worldwide with over 2 million deaths each year. The erythrocyte invasion mechanisms by Plasmodium sp. have been well described, however the physiological aspects involving host components in this process are still poorly understood. Here, we provide evidence for the role of renin-angiotensin system (RAS) components in reducing erythrocyte invasion by P. falciparum. Angiotensin II (Ang II) reduced erythrocyte invasion in an enriched schizont culture of P. falciparum in a dose-dependent manner. Using mass spectroscopy, we showed that Ang II was metabolized by erythrocytes to Ang IV and Ang-(1-7). Parasite infection decreased Ang-(1-7) and completely abolished Ang IV formation. Similar to Ang II, Ang-(1-7) decreased the level of infection in an A779 (specific antagonist of Ang-(1-7) receptor, MAS)-sensitive manner. 10(-7) M PD123319, an AT(2) receptor antagonist, partially reversed the effects of Ang-(1-7) and Ang II. However, 10(-6) M losartan, an antagonist of the AT(1) receptor, had no effect. Gs protein is a crucial player in the Plasmodium falciparum blood cycle and angiotensin peptides can modulate protein kinase A (PKA) activity; 10(-8) M Ang II or 10(-8) M Ang-(1-7) inhibited this activity in erythrocytes by 60% and this effect was reversed by 10(-7) M A779. 10(-6) M dibutyryl-cAMP increased the level of infection and 10(-7) M PKA inhibitor decreased the level of infection by 30%. These results indicate that the effect of Ang-(1-7) on P. falciparum blood stage involves a MAS-mediated PKA inhibition. Our results indicate a crucial role for Ang II conversion into Ang-(1-7) in controlling the erythrocytic cycle of the malaria parasite, adding new functions to peptides initially described to be involved in the regulation of vascular tonus.

Plasmodium falciparum proteome changes in response to doxycycline treatment.

BACKGROUND: The emergence of Plasmodium falciparum resistance to most anti-malarial compounds has highlighted the urgency to develop new drugs and to clarify the mechanisms of anti-malarial drugs currently used. Among them, doxycycline is used alone for malaria chemoprophylaxis or in combination with quinine or artemisinin derivatives for malaria treatment. The molecular mechanisms of doxycycline action in P. falciparum have not yet been clearly defined, particularly at the protein level. METHODS: A proteomic approach was used to analyse protein expression changes in the schizont stage of the malarial parasite P. falciparum following doxycycline treatment. A comparison of protein expression between treated and untreated protein samples was performed using two complementary proteomic approaches: two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and isobaric tagging reagents for relative and absolute quantification (iTRAQ). RESULTS: After doxycycline treatment, 32 and 40 P. falciparum proteins were found to have significantly deregulated expression levels by 2D-DIGE and iTRAQ methods, respectively. Although some of these proteins have been already described as being deregulated by other drug treatments, numerous changes in protein levels seem to be specific to doxycycline treatment, which could perturb apicoplast metabolism. Quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to confirm this hypothesis. CONCLUSIONS: In this study, a specific response to doxycycline treatment was distinguished and seems to involve mitochondrion and apicoplast organelles. These data provide a starting point for the elucidation of drug targets and the discovery of mechanisms of resistance to anti-malarial compounds.

Antimalarial effect of N-acetyl-L-Leucyl-L-leucyl-L-norleucinal by the inhibition of Plasmodium falciparum Calpain.

The biological understanding of malaria parasites has increased considerably over the past two decades with the discovery of many potential targets for the development of new antimalarial drugs. Calpain, a cysteine protease of Plasmodium falciparum, is believed to be a central mediator essential for parasitic activity. However, the utility of calpain as a potential anti-malarial target in P. falciparum has not been fully determined. In the present study, we determined the effect of N-acetyl-L-Leucyl-L-leucyl-L-norleucinal (ALLN)-treatment on the expression of calpain in erythrocytic stages of P. falciparum and its usefulness as an antimalarial chemotherapeutic agent. ALLN was shown to have low toxicity to HeLa cells but high toxicity to malaria. ALLN inhibited the expression of calpain in ring, trophozoite and schizont stages when treated for 48 h. Also, after 48 h, samples were characterized by 6.15% and 0% parasitemia without ALLN treatment and with ALLN treatment, respectively. Brightfield and confocal microscopy revealed that ALLN treatment affects merozoite maturation. As ALLN concentration increased from 1 muM to 100 microM, ring stage parasites did not mature into the schizont stage. When ALLN treatment was continued for 48 h, it also significantly inhibited the maturation of ring-stage parasites into trophozoite or schizont stages and survival of malarial parasites. Taken together, these findings suggest that ALLN inhibit the maturation and survival of P. falciparum and calpain expression, and thus has potential utility as an antimalarial chemotherapeutic agent.

The therapeutic efficacy of sulfadoxine/pyrimethamine against Plasmodium falciparum in Yemen.

OBJECTIVE: The aim of this study was to determine the sensitivities of Plasmodium falciparum clinical isolates to sulfadoxine/pyrimethamine (SP) using in vivo and in vitro methods. SUBJECTS AND METHODS: In vivo and Mark III in-vitro test techniques according to World Health Organization protocols of antimalarial drug tests were used to determine the SP susceptibility of the P. falciparum isolates from 100 malaria patients of both sexes between the ages of 3.5 and 45 years and living in Tihamah, Yemen. The study was conducted between 19 March and 12 May 2005. RESULTS: In vivo: no therapeutic failure occurred; the clinical outcome matched the parasitological response and all patients were parasite free by day 3 and remained so on days 7, 14 and 28. In vitro: all the P. falciparum isolates developed to schizonts in zero-drug-concentration wells, but were inhibited in 40 nmol/l of SP; the mean effective concentration (EC(99)) was 67.17 nmol/l. CONCLUSION: Our findings showed that the SP combination is still effective for the treatment of uncomplicated P. falciparum malaria in Yemen. It is recommended that further studies be carried out to address the importance of dihydropteroate synthetase/dihydrofolate reductase mutations as predictive markers of sulfadoxine/pyrimethamine resistance in Yemen.

Antiplasmodial activity of sesquiterpene lactone from Carpesium rosulatum in mice.

In the previous work, methanol extracts of Carpesium rosulatum (Compositae) were found to have high antiplasmodial activity against Plasmodium falciparum in vitro, this activity being largely attributable to a ineupatorolides A (I-A). In the present study, encouragingly, I-A was also found to have potential antimalarial activity in vivo when tested against Plasmodium berghei in mice. I-A (2, 5, 10 mg kg(-1) day(-1)) exhibited a significant blood schizontocidal activity in 4-day early infection, repository evaluation, and in established infection with a significant mean survival time comparable to that of the standard drug, chloroquine (5 mg kg(-1) day(-1)). The I-A possesses a promising antiplasmodial activity, which can be exploited in malaria therapy.

An antimalarial neem leaf extract has both schizonticidal and gametocytocidal activities.

A crude acetone/water (50/50) extract of neem leaves (IRAB) was evaluated for activity against the asexual (trophozoites/schizonts) and the sexual (gametocytes) forms of the malarial parasite, Plasmodium falciparum, in vitro. In separate 72 hour cultures of both asexual parasites and mature gametocytes treated with IRAB (0.5 microg/mL), parasite numbers were less than 50% of the numbers in control cultures, which had 8.0% and 8.5% parasitemia, respectively. In cultures containing 2.5 microg/mL, asexual parasites and mature and immature gametocytes were reduced to 0.1%, 0.2%, and 0% parasitemia, respectively. There were no parasites in the cultures containing 5.0 microg/mL. This extract, if found safe, may provide materials for development of new antimalarial drugs that may be useful both in treatment of malaria as well as the control of its transmission through gametocytes.