Cytometric quantification of singlet oxygen in the human malaria parasite Plasmodium falciparum

The malaria parasite Plasmodium falciparum proliferates within human erythrocytes and is thereby exposed to a variety of reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radical, superoxide anion, and highly reactive singlet oxygen ((1)O(2)). While most ROS are already well studied in the malaria parasite, singlet oxygen has been neglected to date. In this study we visualized the generation of (1)O(2) by live cell fluorescence microscopy using 3-(p-aminophenyl) fluorescein as an indicator dye. While (1) O(2) is found restrictively in the parasite, its amount varies during erythrocytic schizogony. Since the photosensitizer cercosporin generates defined amounts of (1)O(2) we have established a new cytometric method that allows the stage specific quantification of (1)O(2). Therefore, the parasites were first classified into three main stages according to their respective pixel-area of 200-600 pixels for rings, 700-1,200 pixels for trophozoites and 1,400-2,500 pixels for schizonts. Interestingly the highest mean concentration of endogenous (1)O(2) of 0.34 nM is found in the trophozoites stage, followed by 0.20 nM (ring stage) and 0.10 nM (schizont stage) suggesting that (1)O(2) derives predominantly from the digestion of hemoglobin.     

Plasmodium cynomolgi: immunization of a rhesus monkey with exoerythrocytic stages cultured in autologous hepatocytes

To investigate the immune response to exoerythrocytic stages of malaria parasites, a rhesus monkey was immunized with autologous primary hepatocyte cultures infected with 7-day-old liver stage parasites of Plasmodium cynomolgi. A primary antibody response against EE stage antigens was obtained, and boosted after injection of homologous viable sporozoites. Antibodies directed against sporozoites and blood stages were also detected. The polyvalent immune response observed demonstrates the antigenicity of the liver stages and suggests their involvement in the general immune response against malaria.     

Gametocytocidal activity of pyronaridine and DNA topoisomerase II inhibitors against multidrug-resistant Plasmodium falciparum in vitro

Gametocytocidal activities of pyronaridine and DNA topoisomerase II inhibitors against two isolates of multidrug-resistant Plasmodium falciparum, KT1 and KT3 were determined. After sorbitol treatment, pure gametocyte cultures of Plasmodium falciparum containing mostly young gametocytes (stage II and III) obtained on day 11 were exposed to the drugs for 48 h. The effect of the drugs on gametocyte development was assessed by counting gametocytes on day 15 of culture. Pyronaridine was the most effective gametocytocidal drug against P. falciparum isolates KT1 and KT3 with 50% inhibitory concentration of 6 and 20 nM, respectively. Moreover, the 50% inhibitory concentration of pyronaridine was lower than that of primaquine which is the only drug used to treat malaria patients harboring gametocytes. Prokaryotic (norfloxacin) and eukaryotic (amsacrine and etoposide) DNA topoisomerase II inhibitors were only effective against asexual but not sexual stages of the malaria parasites. Pyronaridine has both schizontocidal and gametocytocidal activities against the human malaria parasite, P. falciparum.     

Activation of a PAK-MEK signalling pathway in malaria parasite-infected erythrocytes

Merozoites of malaria parasites invade red blood cells (RBCs), where they multiply by schizogony, undergoing development through ring, trophozoite and schizont stages that are responsible for malaria pathogenesis. Here, we report that a protein kinase-mediated signalling pathway involving host RBC PAK1 and MEK1, which do not have orthologues in the Plasmodium kinome, is selectively stimulated in Plasmodium falciparum-infected (versus uninfected) RBCs, as determined by the use of phospho-specific antibodies directed against the activated forms of these enzymes. Pharmacological interference with host MEK and PAK function using highly specific allosteric inhibitors in their known cellular IC50 ranges results in parasite death. Furthermore, MEK inhibitors have parasiticidal effects in vitro on hepatocyte and erythrocyte stages of the rodent malaria parasite Plasmodium berghei, indicating conservation of this subversive strategy in malaria parasites. These findings have profound implications for the development of novel strategies for antimalarial chemotherapy.     

Protection from experimental cerebral malaria with a single dose of radiation-attenuated, blood-stage Plasmodium berghei parasites

Background

Whole malaria parasites are highly effective in inducing immunity against malaria. Due to the limited success of subunit based vaccines in clinical studies, there has been a renewed interest in whole parasite-based malaria vaccines. Apart from attenuated sporozoites, there have also been efforts to use live asexual stage parasites as vaccine immunogens.

Methodology and Results

We used radiation exposure to attenuate the highly virulent asexual blood stages of the murine malaria parasite P. berghei to a non-replicable, avirulent form. We tested the ability of the attenuated blood stage parasites to induce immunity to parasitemia and the symptoms of severe malaria disease. Depending on the mouse genetic background, a single high dose immunization without adjuvant protected mice from parasitemia and severe disease (CD1 mice) or from experimental cerebral malaria (ECM) (C57BL/6 mice). A low dose immunization did not protect against parasitemia or severe disease in either model after one or two immunizations. The protection from ECM was associated with a parasite specific antibody response and also with a lower level of splenic parasite-specific IFN-γ production, which is a mediator of ECM pathology in C57BL/6 mice. Surprisingly, there was no difference in the sequestration of CD8+ T cells and CD45+ CD11b+ macrophages in the brains of immunized, ECM-protected mice.

Conclusions

This report further demonstrates the effectiveness of a whole parasite blood-stage vaccine in inducing immunity to malaria and explicitly demonstrates its effectiveness against ECM, the most pathogenic consequence of malaria infection. This experimental model will be important to explore the formulation of whole parasite blood-stage vaccines against malaria and to investigate the immune mechanisms that mediate protection against parasitemia and cerebral malaria.     

Gametocytocidal activity of pyronaridine and DNA topoisomerase II inhibitors against multidrug-resistant Plasmodium falciparum in vitro

Gametocytocidal activities of pyronaridine and DNA topoisomerase II inhibitors against two isolates of multidrug-resistant Plasmodium falciparum, KT1 and KT3 were determined. After sorbitol treatment, pure gametocyte cultures of Plasmodium falciparum containing mostly young gametocytes (stage II and III) obtained on day 11 were exposed to the drugs for 48 h. The effect of the drugs on gametocyte development was assessed by counting gametocytes on day 15 of culture. Pyronaridine was the most effective gametocytocidal drug against P. falciparum isolates KT1 and KT3 with 50% inhibitory concentration of 6 and 20 nM, respectively. Moreover, the 50% inhibitory concentration of pyronaridine was lower than that of primaquine which is the only drug used to treat malaria patients harboring gametocytes. Prokaryotic (norfloxacin) and eukaryotic (amsacrine and etoposide) DNA topoisomerase II inhibitors were only effective against asexual but not sexual stages of the malaria parasites. Pyronaridine has both schizontocidal and gametocytocidal activities against the human malaria parasite, P. falciparum.     

Design of antimalarial transmission blocking agents: Pharmacophore mapping of ligands active against stage-V mature gametocytes of Plasmodium falciparum

Abstract

The discovery of transmission-blocking (T-B) agents is crucial for preventing and complete removal of malaria infection. However, most of the existing antimalarials are only active against the asexual stages of Plasmodium parasite, but ineffective against the sexual stage (gametocytes). In this background, we have developed pharmacophore models against the stage-V mature gametocytes of P. falciparum parasites. The pharmacophore model (Hypo-1) showed five pharmacophoric features namely, one hydrogen bond donor (HBD), one hydrophobic aliphatic (HYAl), one ring aromatic (RA), and two hydrophobic aromatic (HYAr) essential for the anti-gametocytic activity. The amino, methyl, fused phenyl ring of the quinazoline heterocycle, two phenyl rings of biphenyl moiety (HBD, HYAl, HYAr1, HYAr2 and RA) are the crucial features responsible for the non-specific anti-gametocytic activity (PfG). Subsequently, the model (Hypo-2) developed against the stage-V female gametocytes (PffG) showed the contribution of three pharmacophoric features namely, two hydrogen bond acceptor (HYA) and one RA required for the anti-gametocytic activity. The sulfhydryl, imine and pyridyl groups are observed to be essential for anti-gametocytic activity against female gametocytes. Both the models (PfG and PfGG) showed the classification accuracies of 78.26 and 71.64% for training set compounds and 60.80 and 60.18% for the test set compounds, respectively, for classification of compounds into higher and lower active classes. Also, both the models were found to retain the higher active compounds (IC₅₀ <100?nM) in top 1% of total compounds (actives and decoys) as observed after screening the decoy set compounds.

Communicated by Ramaswamy H Sarma     

Metacytofilin has potent anti-malarial activity

Metacytofilin (MCF) was isolated from the fungus Metarhizium sp. TA2759. Although MCF possesses anti-Toxoplasma activity, the effects of this compound against other parasites are unknown. Here, we evaluated the in vitro anti-malarial activity of MCF against the 3D7 strain and the chloroquine-resistant K1 strain of Plasmodium falciparum. The half maximal inhibitory concentrations (IC₅₀) of MCF against the 3D7 and K-1 strains following culture for 48 h were 666 nM and 605 nM, respectively. Artemisinin was more potent than MCF against both strains (3D7 IC₅₀: 17.4 nM; K-1 IC₅₀: 18.3 nM), while chloroquine was ineffective against the chloroquine-resistant strain (3D7 IC₅₀: 39.1 nM; K-1 IC₅₀: 1.62 μM). MCF affected the ring stage of the parasites, resulting in their death as shown by spots within red blood cells. MCF also inhibited parasite growth following culture for 72 h (3D7 IC₅₀, 285 nM). Four optical isomers of cyclo[Leu-Phe]-diketopiperazine derivatives with modified methoxy and/or hydroxyl groups lost anti-malarial activity, suggesting that the spatial positions of the methoxy and hydroxyl groups in MCF play an important role in its anti-malarial effects. Together, these data suggest that MCF may represent a promising lead compound for treatment of drug-resistant malarial parasites.     

A novel ENU-mutation in ankyrin-1 disrupts malaria parasite maturation in red blood cells of mice

The blood stage of the plasmodium parasite life cycle is responsible for the clinical symptoms of malaria. Epidemiological studies have identified coincidental malarial endemicity and multiple red blood cell (RBC) disorders. Many RBC disorders result from mutations in genes encoding cytoskeletal proteins and these are associated with increased protection against malarial infections. However the mechanisms underpinning these genetic, host responses remain obscure. We have performed an N-ethyl-N-nitrosourea (ENU) mutagenesis screen and have identified a novel dominant (haploinsufficient) mutation in the Ank-1 gene (Ank1(MRI23420)) of mice displaying hereditary spherocytosis (HS). Female mice, heterozygous for the Ank-1 mutation showed increased survival to infection by Plasmodium chabaudi adami DS with a concomitant 30% decrease in parasitemia compared to wild-type, isogenic mice (wt). A comparative in vivo red cell invasion and parasite growth assay showed a RBC-autonomous effect characterised by decreased proportion of infected heterozygous RBCs. Within approximately 6-8 hours post-invasion, TUNEL staining of intraerythrocytic parasites, showed a significant increase in dead parasites in heterozygotes. This was especially notable at the ring and trophozoite stages in the blood of infected heterozygous mutant mice compared to wt (p<0.05). We conclude that increased malaria resistance due to ankyrin-1 deficiency is caused by the intraerythrocytic death of P. chabaudi parasites.     

Inhibitory effect of TNF-α on malaria pre-erythrocytic stage development: influence of host hepatocyte/parasite combinations

Background

The liver stages of malaria parasites are inhibited by cytokines such as interferon-γ or Interleukin (IL)-6. Binding of these cytokines to their receptors at the surface of the infected hepatocytes leads to the production of nitric oxide (NO) and radical oxygen intermediates (ROI), which kill hepatic parasites. However, conflicting results were obtained with TNF-α possibly because of differences in the models used. We have reassessed the role of TNF-α in the different cellular systems used to study the Plasmodium pre-erythrocytic stages.

Methods and Findings

Human or mouse TNF-α were tested against human and rodent malaria parasites grown in vitro in human or rodent primary hepatocytes, or in hepatoma cell lines. Our data demonstrated that TNF-α treatment prevents the development of malaria pre-erythrocytic stages. This inhibitory effect however varies with the infecting parasite species and with the nature and origin of the cytokine and hepatocytes. Inhibition was only observed for all parasite species tested when hepatocytes were pre-incubated 24 or 48 hrs before infection and activity was directed only against early hepatic parasite. We further showed that TNF-α inhibition was mediated by a soluble factor present in the supernatant of TNF-α stimulated hepatocytes but it was not related to NO or ROI. Treatment TNF-α prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified.

Conclusions

Treatment TNF-α prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified. However, the nature of the cytokine-host cell-parasite combination must be carefully considered for extrapolation to the human infection.     

The species specificity of immunity generated by live whole organism immunisation with erythrocytic and pre-erythrocytic stages of rodent malaria parasites and implications for vaccine development

A promising strategy for the development of a malaria vaccine involves the use of attenuated whole parasites, as these present a greater repertoire of antigens to the immune system than subunit vaccines. The complexity of the malaria parasite's life cycle offers multiple stages on which to base an attenuated whole organism vaccine. An important consideration in the design and employment of such vaccines is the diversity of the parasites that are infective to humans. The most valuable vaccine would be one that was effective against multiple species/strains of malaria parasite. Here we compare the species specificity of pre-erythrocytic and erythrocytic whole organism vaccination using live parasites with anti-malarial drug attenuation. The cross-stage protection afforded by each vaccination strategy, and the possibility that immunity against one stage may be abrogated by exposure to other stages of both homologous and heterologous parasites was also assessed. The rodent malaria parasites Plasmodium yoelii yoelii and Plasmodium vinckei lentum are to address these questions, as they offer the widest possible genetic distance between sub-species of malaria parasites infectious to rodents. It was found that both erythrocytic and pre-erythrocytic stage immunity generated by live, attenuated parasite vaccination have species-specific components, with pre-erythrocytic stage immunity offering a much broader pan-species protection. We show that the protection achieved following sporozoite inoculation with concurrent mefloquine treatment is almost entirely dependent of CD8(+) T-cells. Evidence is presented for cross-stage protection between erythrocytic and pre-erythrocytic stage vaccination. Finally, it is shown that, with these species, an erythrocytic stage infection of either a homologous or heterologous species following immunisation with pre-erythrocytic stages does not abrogate this immunity. This is the first direct comparison of the specificity and efficacy of erythrocytic and pre-erythrocytic stage whole organism vaccination strategies utilising the same parasite species pair.     

The search for new antimalarial drugs from plants used to treat fever and malaria or plants ramdomly selected: a review.

In this review we discuss the ongoing situation of human malaria in the Brazilian Amazon, where it is endemic causing over 610,000 new acute cases yearly, a number which is on the increase. This is partly a result of drug resistant parasites and new antimalarial drugs are urgently needed. The approaches we have used in the search of new drugs during decades are now reviewed and include ethnopharmocology, plants randomly selected, extracts or isolated substances from plants shown to be active against the blood stage parasites in our previous studies. Emphasis is given on the medicinal plant Bidens pilosa, proven to be active against the parasite blood stages in tests using freshly prepared plant extracts. The anti-sporozoite activity of one plant used in the Brazilian endemic area to prevent malaria is also described, the so called "Indian beer" (Ampelozizyphus amazonicus, Rhamnaceae). Freshly prepared extracts from the roots of this plant were totally inactive against blood stage parasites, but active against sporozoites of Plasmodium gallinaceum or the primary exoerythrocytic stages reducing tissue parasitism in inoculated chickens. This result will be of practical importance if confirmed in mammalian malaria. Problems and perspectives in the search for antimalarial drugs are discussed as well as the toxicological and clinical trials to validate some of the active plants for public health use in Brazil.     

Novel squaramides with in vitro liver stage antiplasmodial activity

A structure–activity relationship study was performed with ten 8-aminoquinoline-squaramides compounds active against liver stage malaria parasites, using human hepatoma cells (Huh7) infected by Plasmodium berghei parasites. In addition, their blood-schizontocidal activity was assessed against chloroquine-resistant W2 strain Plasmodium falciparum. Compound 3 was 7.3-fold more potent than the positive control primaquine against liver-stage parasites, illustrating the importance of the squarate moiety to activity.     

Plasmodium yoelii: activity of azithromycin in combination with pyrimethamine or sulfadoxine against blood and sporozoite induced infections in Swiss mice

The efficacy of pyrimethamine or sulfadoxine administered in combination with azithromycin was examined in a rodent malaria model. Outbred Swiss mice infected with blood stage parasites were treated from day 0 to day 3 and efficacy of different regimens was monitored in terms of the curative response and the delay time to reach 2% parasitaemia (2% DT). Administration of azithromycin alone at 60 mg/kg/day produced curative response while lower doses showed marginally delayed 2% DT. A marked potentiation in activities of pyrimethamine (100-fold) or sulfadoxine (10-fold) was observed when administered at non-curative doses of 0.1 mg/kg/day in combination with azithromycin (30 mg/kg/day) against blood stage parasites. A combination of 10 mg/kg/day azithromycin with 0.3 mg/kg/day sulfadoxine was also curative. Likewise in the causal prophylactic test, a combination regimen comprising 1/16th and 1/3rd the individual curative doses of pyrimethamine and azithromycin, respectively, prevented the development of patent infection after Plasmodium yoelii sporozoite challenge. Our results suggest that a combination of azithromycin with the second line treatment regimen of fansidar may enhance the therapeutic efficacy of the latter and also provide better prophylaxis against Plasmodium falciparum malaria.     

Subpatent infection with nucleoside transporter 1‐deficient Plasmodium blood stage parasites confers sterile protection against lethal malaria in mice

Repeated immunizations with whole Plasmodium blood stage parasites and concomitant drug cure of infection confer protective immunity against parasite challenge in mice, monkeys and humans. Moreover, it was recently shown that infections with genetically modified rodent malaria blood stage parasites conferred sterile protection against lethal blood stage challenge. However, in these models vaccination resulted in high parasitemias and, in consequence, carries risk of vaccine‐induced pathology and death. Herein, we generated a novel, completely blood stage‐attenuated P. yoelii rodent malaria strain by targeted deletion of parasite nucleoside transporter 1 (NT1). Immunization of inbred and outbred mouse strains with a single low dose of Pynt1^‐ blood stages did not induce any patent infections and conferred complete sterile protection against lethal heterologous blood stage and sporozoite challenges. Partial protection was observed against lethal challenges with another parasite species, P. berghei. Importantly, subcutaneous immunization with Pynt1^‐ conferred sterile protection against lethal blood stage challenges. We show that cellular and humoral immune responses are both essential for sterile protection. The study demonstrates that genetic manipulation provides a platform for the designed, complete attenuation of malaria parasite blood stages and suggests testing the safety and efficacy of P. falciparum NT1 knockout strains in humans.     

Fluorescent Plasmodium berghei sporozoites and pre-erythrocytic stages: a new tool to study mosquito and mammalian host interactions with malaria parasites

To track malaria parasites for biological studies within the mosquito and mammalian hosts, we constructed a stably transformed clonal line of Plasmodium berghei, PbFluspo, in which sporogonic and pre‐erythrocytic liver‐stage parasites are autonomously fluorescent. A cassette containing the structural gene for the FACS‐adapted green fluorescent protein mutant 2 (GFPmut2), expressed from the 5′ and 3′ flanking sequences of the circumsporozoite (CS) protein gene, was integrated and expressed at the endogenous CS locus. Recombinant parasites, which bear a wild‐type copy of CS, generated highly fluorescent oocysts and sporozoites that invaded mosquito salivary glands and were transmitted normally to rodent hosts. The parasites infected cultured hepatocytes in vitro, where they developed into fluorescent pre‐erythrocytic forms. Mammalian cells infected by these parasites can be separated from non‐infected cells by fluorescence activated cell sorter (FACS) analysis. These fluorescent insect and mammalian stages of P. berghei should be useful for phenotypic studies in their respective hosts, as well as for identification of new genes expressed in these parasite stages.     

Synthesis, in vitro antimalarial activities and cytotoxicities of amino-artemisinin-ferrocene derivatives

Novel derivatives bearing a ferrocene attached via a piperazine linker to C-10 of the artemisinin nucleus were prepared from dihydroartemisinin and screened against chloroquine (CQ) sensitive NF54 and CQ resistant K1 and W2 strains of Plasmodium falciparum (Pf) parasites. The overall aim is to imprint oxidant (from the artemisinin) and redox (from the ferrocene) activities. In a preliminary assessment, these compounds were shown to possess activities in the low nM range with the most active being compound 6 with IC₅₀ values of 2.79?nM against Pf K1 and 3.2?nM against Pf W2. Overall the resistance indices indicate that the compounds have a low potential for cross resistance. Cytotoxicities were determined with Hek293 human embryonic kidney cells and activities against proliferating cells were assessed against A375 human malignant melanoma cells. The selectivity indices of the amino-artemisinin ferrocene derivatives indicate there is overall an appreciably higher selectivity towards the malaria parasite than mammalian cells.     

Blockage of spontaneous Ca2+ oscillation causes cell death in intraerythrocitic Plasmodium falciparum

Malaria remains one of the world's most important infectious diseases and is responsible for enormous mortality and morbidity. Resistance to antimalarial drugs is a challenging problem in malaria control. Clinical malaria is associated with the proliferation and development of Plasmodium parasites in human erythrocytes. Especially, the development into the mature forms (trophozoite and schizont) of Plasmodium falciparum (P. falciparum) causes severe malaria symptoms due to a distinctive property, sequestration which is not shared by any other human malaria. Ca(2+) is well known to be a highly versatile intracellular messenger that regulates many different cellular processes. Cytosolic Ca(2+) increases evoked by extracellular stimuli are often observed in the form of oscillating Ca(2+) spikes (Ca(2+) oscillation) in eukaryotic cells. However, in lower eukaryotic and plant cells the physiological roles and the molecular mechanisms of Ca(2+) oscillation are poorly understood. Here, we showed the observation of the inositol 1,4,5-trisphospate (IP(3))-dependent spontaneous Ca(2+) oscillation in P. falciparum without any exogenous extracellular stimulation by using live cell fluorescence Ca(2+) imaging. Intraerythrocytic P. falciparum exhibited stage-specific Ca(2+) oscillations in ring form and trophozoite stages which were blocked by IP(3) receptor inhibitor, 2-aminoethyl diphenylborinate (2-APB). Analyses of parasitaemia and parasite size and electron micrograph of 2-APB-treated P. falciparum revealed that 2-APB severely obstructed the intraerythrocytic maturation, resulting in cell death of the parasites. Furthermore, we confirmed the similar lethal effect of 2-APB on the chloroquine-resistant strain of P. falciparum. To our best knowledge, we for the first time showed the existence of the spontaneous Ca(2+) oscillation in Plasmodium species and clearly demonstrated that IP(3)-dependent spontaneous Ca(2+) oscillation in P. falciparum is critical for the development of the blood stage of the parasites. Our results provide a novel concept that IP(3)/Ca(2+) signaling pathway in the intraerythrocytic malaria parasites is a promising target for antimalarial drug development.