Plasmodium Life Cycle Research Articles

EWGWS insert in Plasmodium falciparum ookinete surface enolase is involved in binding of PWWP containing peptides: Implications to mosquito midgut invasion by the parasite

There are multiple stages in the life cycle of Plasmodium that invade host cells. Molecular machinery involved is such host–pathogen interactions constitute excellent drug targets and/or vaccine candidates. A screen using a phage display library has previously demonstrated presence of enolase on the surface of the Plasmodium ookinete. Phage-displayed peptides that bound to the ookinete contained a conserved motif (PWWP) in their sequence. Here, direct binding of these peptides with recombinant Plasmodium falciparum enolase (rPfeno) was investigated. These peptides showed specific binding to rPfeno, but failed to bind to other enolases. Plasmodium spp enolases are distinct in having an insert of five amino acids (104EWGWS108) that is not found in host enolases. The possibility of this insert being the recognition motif for the PWWP containing peptides was examined, (i) by comparing the binding of the peptides with rPfeno and a deletion variant Δ-rPfeno lacking 104EWGWS108, (ii) by measuring the changes in proton chemical shifts of PWWP peptides on binding to different enolases and (iii) by inter-molecular docking experiment to locate the peptide binding site. Results from these studies showed that the pentapeptide insert of Pfeno indeed constitutes the binding site for the PWWP domain containing peptide ligands. Search for sequences homologous to phage displayed peptides among peritrophic matrix proteins resulted in identification of perlecan, laminin, peritrophin and spacran. The possibility of these PWWP domain-containing proteins in the peritrophic matrix of insect gut to interact with ookinete cell surface enolase and facilitate the invasion of mosquito midgut epithelium is discussed.

Vaccine approaches to malaria control and elimination: Insights from mathematical models

A licensed malaria vaccine would provide a valuable new tool for malaria control and elimination efforts. Several candidate vaccines targeting different stages of the malaria parasite's lifecycle are currently under development, with one candidate, RTS,S/AS01 for the prevention of Plasmodium falciparum infection, having recently completed Phase III trials. Predicting the public health impact of a candidate malaria vaccine requires using clinical trial data to estimate the vaccine's efficacy profile—the initial efficacy following vaccination and the pattern of waning of efficacy over time. With an estimated vaccine efficacy profile, the effects of vaccination on malaria transmission can be simulated with the aid of mathematical models.Here, we provide an overview of methods for estimating the vaccine efficacy profiles of pre-erythrocytic vaccines and transmission-blocking vaccines from clinical trial data. In the case of RTS,S/AS01, model estimates from Phase II clinical trial data indicate a bi-phasic exponential profile of efficacy against infection, with efficacy waning rapidly in the first 6 months after vaccination followed by a slower rate of waning over the next 4 years. Transmission-blocking vaccines have yet to be tested in large-scale Phase II or Phase III clinical trials so we review ongoing work investigating how a clinical trial might be designed to ensure that vaccine efficacy can be estimated with sufficient statistical power. Finally, we demonstrate how parameters estimated from clinical trials can be used to predict the impact of vaccination campaigns on malaria using a mathematical model of malaria transmission.

The role of a parasite-specific D-site in activation of Plasmodium falciparum cGMP-dependent protein kinase

Background Malaria is one of the most dangerous tropical diseases worldwide, resulting in approximately 1.5-2.7 million deaths per year [1]. Furthermore, malaria belongs to the four major infectious diseases also including HIV, tuberculosis and hepatitis. In humans, malaria is transmitted by four species of the genus Plasmodium. However, most malaria deaths are caused by Plasmodium falciparum [2]. The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is one of the key regulators of the malaria parasite life cycle in both sexual and asexual blood-stages. Inhibition of PfPKG stops differentiation and transmission of the parasites, indicating that this kinase is a promising drug target for malaria [3-5]. However, despite its physiological importance, the activation mechanism of PfPKG is not fully understood. Recently, our group discovered that disrupting cGMP binding at the C-terminal cyclic nucleotide-binding (CNB-D) domain almost completely abolishes the kinase activation [6]. Therefore, we investigate the functional role of the PfCNB-D in PfPKG activation.

Editorial: Breaking the cycle: attacking the malaria parasite in the liver.

Editorial: Breaking the cycle: attacking the malaria parasite in the liver.

Plasmodial HSP70s are functionally adapted to the malaria parasite life cycle.

The human malaria parasite, Plasmodium falciparum, encodes a minimal complement of six heat shock protein 70s (PfHSP70s), some of which are highly expressed and are thought to play an important role in the survival and pathology of the parasite. In addition to canonical features of molecular chaperones, these HSP70s possess properties that reflect functional adaptation to a parasitic life style, including resistance to thermal insult during fever periods and host–parasite interactions. The parasite even exports an HSP70 to the host cell where it is likely to be involved in host cell modification. This review focuses on the features of the PfHSP70s, particularly with respect to their adaptation to the malaria parasite life cycle.

Conditional Degradation of Plasmodium Calcineurin Reveals Functions in Parasite Colonization of both Host and Vector.

SummaryFunctional analysis of essential genes in the malarial parasite, Plasmodium, is hindered by lack of efficient strategies for conditional protein regulation. We report the development of a rapid, specific, and inducible chemical-genetic tool in the rodent malaria parasite, P. berghei, in which endogenous proteins engineered to contain the auxin-inducible degron (AID) are selectively degraded upon adding auxin. Application of AID to the calcium-regulated protein phosphatase, calcineurin, revealed functions in host and vector stages of parasite development. Whereas depletion of calcineurin in late-stage schizonts demonstrated its critical role in erythrocyte attachment and invasion in vivo, stage-specific depletion uncovered roles in gamete development, fertilization, and ookinete-to-oocyst and sporozoite-to-liver stage transitions. Furthermore, AID technology facilitated concurrent generation and phenotyping of transgenic lines, allowing multiple lines to be assessed simultaneously with significant reductions in animal use. This study highlights the broad applicability of AID for functional analysis of proteins across the Plasmodium life cycle.

L-Arginine supplementation in mice enhances NO production in spleen cells and inhibits Plasmodium yoelii transmission in mosquitoes.

BackgroundThe life cycle of Plasmodium is complex, requiring invasion of two different hosts, humans and mosquitoes. In humans, initiation of an effective Th1 response during early infection is critical for the control of parasite multiplication. In mosquitoes, inhibition of the development of sexual-stage parasites interrupts the parasite transmission. In this study, we aim to investigate whether dietary supplementation of L-arginine (L-Arg) in mice affects Plasmodium yoelii 17XL (Py17XL) transmission in mosquitoes.MethodsBALB/c mice were orally administered with 1.5 mg/g L-Arg daily for 7 days and infected with Py17XL. The mRNA levels of inducible nitric oxide synthase (iNOS) and arginase 1 in spleen cells were determined by real-time RT-PCR. The amount of nitric oxide (NO) released by spleen cells in vitro was determined by the Griess method. The effect of L-Arg supplementation on subsequent development of P. yoelii gametocytes was evaluated by an in vitro ookinete culture assay and mosquito feeding assay.ResultsPretreatment of mice with L-Arg significantly increased the transcript level of iNOS in spleen cells and the amount of NO synthesized. Dietary L-Arg supplementation also significantly reduced the number of zygotes and ookinetes formed during in vitro culture and the number of oocysts formed on mosquito midguts after blood feeding.ConclusionsL-Arg enhances host immunity against blood-stage parasites as well as suppressing subsequent parasite development in mosquitoes. L-Arg as an inexpensive and safe supplement may be used as a novel adjunct treatment against malarial infection.

Tyrosine kinase inhibitors: New class of antimalarials on the horizon?

Development of the antimalarial drug resistant strains has currently become a major public health challenge. There is an urgent need to develop new antimalarial drugs. Tyrosine kinase inhibitors (TKIs) are receiving increasing attention as anticancer therapy. It has revolutionarised the management of CML to say the least. TKIs are also increasingly being implicated in complicated but vital life cycle of malaria parasite. Hence we tested two commonly used but different classes of TKIs (imatinib and sorafenib) in-vitro for their antimalarial activity and possible synergistic activity with existing antimalarial drug. Antimalarial activity was tested with the help of modified WHO microtest technique in-vitro for five different Plasmodium falciparum laboratory strains (3D7, Dd2, 7G8, MRC2, PKL9). Imatinib and sorafenib showed a promising antimalarial activity with all the strains. These compounds caused dose dependent inhibition of parasite maturation. The isobologram analysis of the interactions of these TKIs with standard antimalarial drug, artesunate revealed distinct patterns of synergism, additivity and antagonism at different ratios. Imatinib showed worthwhile synergism with artesunate indicating imatinib and other tyrosine kinase inhibitors may have significant antimalarial activity and can be used in combination therapy.

Mechanisms of hematin crystallization and inhibition by the antimalarial drug chloroquine

Hematin crystallization is the primary mechanism of heme detoxification in malaria parasites and the target of the quinoline class of antimalarials. Despite numerous studies of malaria pathophysiology, fundamental questions regarding hematin growth and inhibition remain. Among them are the identity of the crystallization medium in vivo, aqueous or organic; the mechanism of crystallization, classical or nonclassical; and whether quinoline antimalarials inhibit crystallization by sequestering hematin in the solution, or by blocking surface sites crucial for growth. Here we use time-resolved in situ atomic force microscopy (AFM) and show that the lipid subphase in the parasite may be a preferred growth medium. We provide, to our knowledge, the first evidence of the molecular mechanisms of hematin crystallization and inhibition by chloroquine, a common quinoline antimalarial drug. AFM observations demonstrate that crystallization strictly follows a classical mechanism wherein new crystal layers are generated by 2D nucleation and grow by the attachment of solute molecules. We identify four classes of surface sites available for binding of potential drugs and propose respective mechanisms of drug action. Further studies reveal that chloroquine inhibits hematin crystallization by binding to molecularly flat {100} surfaces. A 2-μM concentration of chloroquine fully arrests layer generation and step advancement, which is ∼10(4)× less than hematin's physiological concentration. Our results suggest that adsorption at specific growth sites may be a general mode of hemozoin growth inhibition for the quinoline antimalarials. Because the atomic structures of the identified sites are known, this insight could advance the future design and/or optimization of new antimalarials.

Crystal structures of the carboxyl cGMP binding domain of the Plasmodium falciparum cGMP-dependent protein kinase reveal a novel capping triad crucial for merozoite egress.

The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is a key regulator across the malaria parasite life cycle. Little is known about PfPKG’s activation mechanism. Here we report that the carboxyl cyclic nucleotide binding domain functions as a “gatekeeper” for activation by providing the highest cGMP affinity and selectivity. To understand the mechanism, we have solved its crystal structures with and without cGMP at 2.0 and 1.9 Å, respectively. These structures revealed a PfPKG-specific capping triad that forms upon cGMP binding, and disrupting the triad reduces kinase activity by 90%. Furthermore, mutating these residues in the parasite prevents blood stage merozoite egress, confirming the essential nature of the triad in the parasite. We propose a mechanism of activation where cGMP binding allosterically triggers the conformational change at the αC-helix, which bridges the regulatory and catalytic domains, causing the capping triad to form and stabilize the active conformation.

An overview of malaria transmission from the perspective of Amazon Anopheles vectors.

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.

Hemozoin activates the innate immune system and reduces Plasmodium berghei infection in Anopheles gambiae.

BackgroundMalaria is a worldwide infectious disease caused by Plasmodium parasites and transmitted by female Anopheles mosquitoes. The malaria vector mosquito Anopheles can trigger effective mechanisms to control completion of the Plasmodium lifecycle; the mosquito immune response to the parasite involves several pathways which are not yet well characterized. Plasmodium metabolite hemozoin has emerged as a potent immunostimulator of mammalian tissues. In this study, we aim to investigate the role of this parasite’s by-product as stimulator of Anopheles gambiae immunity to Plasmodium berghei.MethodsFemale mosquitoes were inoculated with hemozoin and the Plasmodium infection rate and intensity were measured. Differences between treatments were detected by Zero-inflated models. Microarray transcription analysis was performed to assess gene expression response to hemozoin. Genome-wide analysis results were confirmed by stimulation of Anopheles gambiae tissues and cells with hemozoin and silencing of REL2-F and its negative regulator Caspar.ResultsGene expression profiles revealed that hemozoin activates several immunity genes, including pattern recognition receptors (PRRs) and antimicrobial peptides (AMPs). Importantly, we found that the Immune deficiency (Imd) pathway Nuclear Factor-kappaB (NF-κB) transcription factor REL2, in its full-length form REL2-F, was induced upon hemozoin treatment.ConclusionsWe have for the first time shown the impact of hemozoin treatment in Plasmodium infection, reducing both rate and intensity of the infection. We propose that hemozoin boosts the innate immunity in Anopheles, activating key effector genes involved in mosquito resistance to Plasmodium, and this activation is REL2-mediated.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-014-0619-y) contains supplementary material, which is available to authorized users.

Investigation of antimalarial activity, cytotoxicity and action mechanism of piperazine derivatives of betulinic acid.

To semisynthesise piperazine derivatives of betulinic acid to evaluate antimalarial activity, cytotoxicity and action mechanism. The new derivatives were evaluated against the CQ-sensitive Plasmodium falciparum 3D7 strain by flow cytometry (FC) using YOYO-1 as stain. Cytotoxicity of 4a and 4b was performed with HEK293T cells for 24 and 48 h by MTT assay. The capability of compound 4a to modulate Ca(2+) in the trophozoite stage was investigated. The trophozoites were stained with Fluo4-AM and analysed by spectrofluorimetry. Effect on mitochondrial membrane potential (ΔΨm) was tested for 4a by FC with DiOC6 (3) as stain. For β-haematin assay, 4a was incubated for 24 h with reagents such as haemin, and the fluorescence was measured by FlexStation at an absorbance of 405 nm. Antimalarial activity of 4a and 4b was IC50 = 1 and 4 μm, respectively. Compound 4a displayed cytotoxicity with IC50 = 69 and 29 μm for 24 and 48 h, respectively, and 4b was not cytotoxic at the tested concentrations. Addition of 4a leads to an increase in cytosolic Ca(2+) . We have measured ΔΨm after treating parasites with the compound. Data on Figure 4a show that mitochondria were not affected. The action mechanism for 4a, inhibition of β-haematin formation (17%), was lower than CQ treatment (83%; IC50 = 3 mm). Compound 4a showed excellent antimalarial activity, and its action mechanism is involved in Ca(2+) pathway(s).

The sound of sexual commitment breaks the silencing of malaria parasites

A fundamental binary decision is made by malaria parasites at every asexual cycle in the blood between further proliferation and differentiation into gametocytes, the mosquito transmissible stages. Recent studies on Plasmodium epigenetic regulation, transcriptional control and genetic basis of gametocyte production are merging today to unveil players and propose molecular mechanisms of this key branch point in the malaria parasite life cycle.

Synthesis, and anti-malarial screening, of 1-diethylamino-4- (dihydroartemisinin-10-yl)amino pentane

Artemisinin and its derivatives have become antimalarial drugs of choice because they are effective against most stages in the life cycle of plasmodium and are safe for all, including pregnant women. World Health Organisation (WHO) has nevertheless recommended artemisinin based combination therapy (ACT) to guard against possible development of resistance, as observed with chloroquine. In this study we coupled dihydroartemisinin with 2-amino- 5-diethyl aminopentane (the chloroquine handle) by using the Mitsunobu coupling (an SN2 reaction). This involves the use of diisopropylazodicarboxylate (DIAD), triphenyl phosphine (Ph3P), 2-amino-5-diethylaminopentane and dihydroartemisinin (DHA) at room temperature to synthesize the target compound, 1-diethylamino-4- (dihydroartemisinin-10-yl)amino pentane {coded: DHA-CQ; IUPAC Name: N1,N1-diethyl-N4-(3,6,9- trimethyldecahydro- 3,12-epoxy[1,2]dioxepino[4,3-i] isochromen-10-yl) pentane-1,4-diamine}. The structure of DHA-CQ was determined from spectroscopic data (IR, 1H & 13C NMR, MS). The compound was screened at three dose levels of 3 mg/kg, 10 mg/kg and 30 mg/kg, for in vivo curative antimalarial activity against mice infected with Plasmodium berghei berghei. The target compound also had an LD50 of 330 mg/kg in mice by the oral route. A single dose kinetics study was carried out and three different metabolites were identified. Keywords : Artemisinin; Chloroquine; Dihydroartemisinin; Plasmodium berghei berghei; Mitsunobu coupling

Immunization against a merozoite sheddase promotes multiple invasion of red blood cells and attenuates Plasmodium infection in mice.

BackgroundSubtilisin-like protease 2 (SUB2) is a conserved serine protease utilized by Plasmodium parasites as a surface sheddase required for successful merozoite invasion of host red blood cells and has been implicated in ookinete invasion of the mosquito midgut. To determine if SUB2 is a suitable vaccine target to interfere with malaria parasite development, the effects of SUB2-immunization on the Plasmodium life cycle were examined in its vertebrate and invertebrate hosts.MethodsSwiss Webster mice were immunized with SUB2 peptides conjugated to Keyhole limpet hemocyanin (KLH) or KLH alone, and then challenged with Plasmodium berghei. To determine the effects of immunization on parasite development, infected mice were evaluated by blood film and Giemsa staining. In addition, collected immune sera were used to perform passive immunization experiments in non-immunized, P. berghei-infected mice to determine the potential role of SUB2 in parasite development in the mosquito.ResultsFollowing P. berghei challenge, SUB2-immunized mice develop a lower parasitaemia and show improved survival when compared to control immunized mice. Moreover, SUB2 immunization results in an increase in the number of multiply invaded red blood cells, suggesting that SUB2 antibodies interfere with merozoite invasion. Passive immunization experiments imply that SUB2 may not have a major role in ookinete invasion, but this requires further investigation.ConclusionBy interfering with red blood cell invasion, immunization against SUB2 limits malaria parasite development and confers protection from severe malaria. Together, these results provide proof-of-principle evidence for future investigation into the use of SUB2 as a vaccine or drug target to interrupt parasite development in more relevant human malaria models.Electronic supplementary materialThe online version of this article (doi:10.1186/1475-2875-13-313) contains supplementary material, which is available to authorized users.

Exploiting the coenzyme A biosynthesis pathway for the identification of new antimalarial agents: the case for pantothenamides.

Malaria kills more than half a million people each year. There is no vaccine, and recent reports suggest that resistance is developing to the antimalarial regimes currently recommended by the World Health Organization. New drugs are therefore needed to ensure malaria treatment options continue to be available. The intra-erythrocytic stage of the malaria parasite's life cycle is dependent on an extracellular supply of pantothenate (vitamin B5), the precursor of CoA (coenzyme A). It has been known for many years that proliferation of the parasite during this stage of its life cycle can be inhibited with pantothenate analogues. We have shown recently that pantothenamides, a class of pantothenate analogues with antibacterial activity, inhibit parasite proliferation at submicromolar concentrations and do so competitively with pantothenate. These compounds, however, are degraded, and therefore rendered inactive, by the enzyme pantetheinase (vanin), which is present in serum. In the present mini-review, we discuss the two strategies that have been put forward to overcome pantetheinase-mediated degradation of pantothenamides. The strategies effectively provide an opportunity for pantothenamides to be tested in vivo. We also put forward our 'blueprint' for the further development of pantothenamides (and other pantothenate analogues) as potential antimalarials.

Metabolomics in the fight against malaria.

Metabolomics uses high-resolution mass spectrometry to provide a chemical fingerprint of thousands of metabolites present in cells, tissues or body fluids. Such metabolic phenotyping has been successfully used to study various biologic processes and disease states. High-resolution metabolomics can shed new light on the intricacies of host-parasite interactions in each stage of the Plasmodium life cycle and the downstream ramifications on the host’s metabolism, pathogenesis and disease. Such data can become integrated with other large datasets generated using top-down systems biology approaches and be utilised by computational biologists to develop and enhance models of malaria pathogenesis relevant for identifying new drug targets or intervention strategies. Here, we focus on the promise of metabolomics to complement systems biology approaches in the quest for novel interventions in the fight against malaria. We introduce the Malaria Host-Pathogen Interaction Center (MaHPIC), a new systems biology research coalition. A primary goal of the MaHPIC is to generate systems biology datasets relating to human and non-human primate (NHP) malaria parasites and their hosts making these openly available from an online relational database. Metabolomic data from NHP infections and clinical malaria infections from around the world will comprise a unique global resource.

Plasmodium yoelii vitamin B5 pantothenate transporter candidate is essential for parasite transmission to the mosquito.

In nearly all non-photosynthetic cells, pantothenate (vitamin B5) transport and utilization are prerequisites for the synthesis of the universal essential cofactor Coenzyme A (CoA). Early studies showed that human malaria parasites rely on the uptake of pantothenate across the parasite plasma membrane for survival within erythrocytes. Recently, a P. falciparum candidate pantothenate transporter (PAT) was characterized by functional complementation in yeast. These studies revealed that PfPAT mediated survival of yeast cells in low pantothenate concentrations and restored sensitivity of yeast cells lacking pantothenate uptake to fenpropimorph. In addition, PfPAT was refractory to deletion in P. falciparum in vitro, but nothing is known about the in vivo functions of PAT in Plasmodium life cycle stages. Herein, we used gene-targeting techniques to delete PAT in Plasmodium yoelii. Parasites lacking PAT displayed normal asexual and sexual blood stage development compared to wild-type (WT) and WT-like p230p(-) parasites. However, progression from the ookinete to the oocyst stage and sporozoite formation were completely abolished in pat(-) parasites. These studies provide the first evidence for an essential role of a candidate pantothenate transport in malaria transmission to Anopheles mosquitoes. This will set the stage for the development of PAT inhibitors against multiple parasite life cycle stages.

Plasmodium falciparum signal peptide peptidase cleaves malaria heat shock protein 101 (HSP101). Implications for gametocytogenesis

Previously we described the identification of a Plasmodium falciparum signal peptide peptidase (PfSPP) functioning at the blood stage of malaria infection. Our studies also demonstrated that mammalian SPP inhibitors prevent malaria parasite growth at the late-ring/early trophozoite stage of intra-erythrocytic development. Consistent with its role in development, we tested the hypothesis that PfSPP functions at the endoplasmic reticulum of P.falciparum where it cleaves membrane-bound signal peptides generated following the enzyme activity of signal peptidase. The localization of PfSPP to the endoplasmic reticulum was confirmed by immunofluorescence microscopy and immunogold electron microscopy. Biochemical analysis indicated the existence of monomer and dimer forms of PfSPP in the parasite lysate. A comprehensive bioinformatics screen identified several candidate PfSPP substrates in the parasite genome. Using an established transfection based in vivo luminescence assay, malaria heat shock protein 101 (HSP101) was identified as a substrate of PfSPP, and partial inhibition of PfSPP correlated with the emergence of gametocytes. This finding unveils the first known substrate of PfSPP, and provides new perspectives for the function of intra-membrane proteolysis at the erythrocyte stage of malaria parasite life cycle.