Malaria Parasite Plasmodium Falciparum Research Articles

Glycosphingolipid GM3 is localized in both exoplasmic and cytoplasmic leaflets of Plasmodium falciparum malaria parasite plasma membrane

Lipid rafts, sterol-rich and sphingolipid-rich microdomains on the plasma membrane are important in processes like cell signaling, adhesion, and protein and lipid transport. The virulence of many eukaryotic parasites is related to raft microdomains on the cell membrane. In the malaria parasite Plasmodium falciparum, glycosylphosphatidylinositol-anchored proteins, which are important for invasion and are possible targets for vaccine development, are localized in the raft. However, rafts are poorly understood. We used quick-freezing and freeze-fracture immuno-electron microscopy to examine the localization of monosialotetrahexosylganglioside (GM1) and monosialodihexosylganglioside (GM3), putative raft microdomain components in P. falciparum and infected erythrocytes. This method immobilizes molecules in situ, minimizing artifacts. GM3 was localized in the exoplasmic (EF) and cytoplasmic leaflets (PF) of the parasite and the parasitophorous vacuole (PV) membranes, but solely in the EF of the infected erythrocyte membrane, as in the case for uninfected erythrocytes. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) was localized solely in the PF of erythrocyte, parasite, and PV membranes. This is the first time that GM3, the major component of raft microdomains, was found in the PF of a biological membrane. The unique localization of raft microdomains may be due to P. falciparum lipid metabolism and its unique biological processes, like protein transport from the parasite to infected erythrocytes.

Plasmodium falciparum K13 mutations in Africa and Asia impact artemisinin resistance and parasite fitness.

The emergence of mutant K13-mediated artemisinin (ART) resistance in Plasmodium falciparum malaria parasites has led to widespread treatment failures across Southeast Asia. In Africa, K13-propeller genotyping confirms the emergence of the R561H mutation in Rwanda and highlights the continuing dominance of wild-type K13 elsewhere. Using gene editing, we show that R561H, along with C580Y and M579I, confer elevated in vitro ART resistance in some African strains, contrasting with minimal changes in ART susceptibility in others. C580Y and M579I cause substantial fitness costs, which may slow their dissemination in high-transmission settings, in contrast with R561H that in African 3D7 parasites is fitness neutral. In Cambodia, K13 genotyping highlights the increasing spatio-temporal dominance of C580Y. Editing multiple K13 mutations into a panel of Southeast Asian strains reveals that only the R561H variant yields ART resistance comparable to C580Y. In Asian Dd2 parasites C580Y shows no fitness cost, in contrast with most other K13 mutations tested, including R561H. Editing of point mutations in ferredoxin or mdr2, earlier associated with resistance, has no impact on ART susceptibility or parasite fitness. These data underline the complex interplay between K13 mutations, parasite survival, growth and genetic background in contributing to the spread of ART resistance.

Atomic Resolution Homology Models and Molecular Dynamics Simulations of Plasmodium falciparum Tubulins.

Microtubules are tubulin polymers present in the eukaryotic cytoskeleton essential for structural stability and cell division that are also roadways for intracellular transport of vesicles and organelles. In the human malaria parasite Plasmodium falciparum, apart from providing structural stability and cell division, microtubules also facilitate important biological activities crucial for parasite survival in hosts, such as egression and motility. Hence, parasite structures and processes involving microtubules are among the most important drug targets for discovering much-needed novel Plasmodium inhibitors. The current study aims to construct reliable and high-quality 3D models of α-, β-, and γ-tubulins using various modeling techniques. We identified a common binding pocket specific to Plasmodium α-, β-, and γ-tubulins. Molecular dynamics simulations confirmed the stability of the Plasmodium tubulin 3D structures. The models generated in the present study may be used for protein–protein and protein–drug interaction investigations targeted toward designing malaria parasite tubulin-specific inhibitors.

The Transcription Factor PfAP2-O Influences Virulence Gene Transcription and Sexual Development in Plasmodium falciparum

The human malaria parasite Plasmodium falciparum expresses variant PfEMP1 proteins on the infected erythrocyte, which function as ligands for endothelial receptors in capillary vessels, leading to erythrocyte sequestration and severe malaria. The factors that orchestrate the mono-allelic expression of the 45–90 PfEMP1-encoding var genes within each parasite genome are still not fully identified. Here, we show that the transcription factor PfAP2-O influences the transcription of var genes. The temporary knockdown of PfAP2-O leads to a complete loss of var transcriptional memory and a decrease in cytoadherence in CD36 adherent parasites. AP2-O-knocked-down parasites exhibited also significant reductions in transmission through Anopheles mosquitoes. We propose that PfAP2-O is, beside its role in transmission stages, also one of the virulence gene transcriptional regulators and may therefore be exploited as an important target to disrupt severe malaria and block parasite transmission.

The power and promise of genetic mapping from Plasmodium falciparum crosses utilizing human liver-chimeric mice

Genetic crosses are most powerful for linkage analysis when progeny numbers are high, parental alleles segregate evenly and numbers of inbred progeny are minimized. We previously developed a novel genetic crossing platform for the human malaria parasite Plasmodium falciparum, an obligately sexual, hermaphroditic protozoan, using mice carrying human hepatocytes (the human liver-chimeric FRG NOD huHep mouse) as the vertebrate host. We report on two genetic crosses—(1) an allopatric cross between a laboratory-adapted parasite (NF54) of African origin and a recently patient-derived Asian parasite, and (2) a sympatric cross between two recently patient-derived Asian parasites. We generated 144 unique recombinant clones from the two crosses, doubling the number of unique recombinant progeny generated in the previous 30 years. The allopatric African/Asian cross has minimal levels of inbreeding and extreme segregation distortion, while in the sympatric Asian cross, inbred progeny predominate and parental alleles segregate evenly. Using simulations, we demonstrate that these progeny provide the power to map small-effect mutations and epistatic interactions. The segregation distortion in the allopatric cross slightly erodes power to detect linkage in several genome regions. We greatly increase the power and the precision to map biomedically important traits with these new large progeny panels.

Metabolome modulation of the host adaptive immunity in human malaria.

Host responses to infection with the malaria parasite Plasmodium falciparum vary among individuals for reasons that are poorly understood. Here we reveal metabolic perturbations as a consequence of malaria infection in children and identify an immunosuppressive role of endogenous steroid production in the context of P. falciparum infection. We perform metabolomics on matched samples from children from two ethnic groups in West Africa, before and after infection with seasonal malaria. Analysing 306 global metabolomes, we identify 92 parasitaemia-associated metabolites with impact on the host adaptive immune response. Integrative metabolomic and transcriptomic analyses, and causal mediation and moderation analyses, reveal an infection-driven immunosuppressive role of parasitaemia-associated pregnenolone steroids on lymphocyte function and the expression of key immunoregulatory lymphocyte genes in the Gouin ethnic group. In children from the less malaria-susceptible Fulani ethnic group, we observe opposing responses following infection, consistent with the immunosuppressive role of endogenous steroids in malaria. These findings advance our understanding of P. falciparum pathogenesis in humans and identify potential new targets for antimalarial therapeutic interventions.

Sialic Acids as Receptors for Pathogens.

Carbohydrates have long been known to mediate intracellular interactions, whether within one organism or between different organisms. Sialic acids (Sias) are carbohydrates that usually occupy the terminal positions in longer carbohydrate chains, which makes them common recognition targets mediating these interactions. In this review, we summarize the knowledge about animal disease-causing agents such as viruses, bacteria and protozoa (including the malaria parasite Plasmodium falciparum) in which Sias play a role in infection biology. While Sias may promote binding of, e.g., influenza viruses and SV40, they act as decoys for betacoronaviruses. The presence of two common forms of Sias, Neu5Ac and Neu5Gc, is species-specific, and in humans, the enzyme converting Neu5Ac to Neu5Gc (CMAH, CMP-Neu5Ac hydroxylase) is lost, most likely due to adaptation to pathogen regimes; we discuss the research about the influence of malaria on this trait. In addition, we present data suggesting the CMAH gene was probably present in the ancestor of animals, shedding light on its glycobiology. We predict that a better understanding of the role of Sias in disease vectors would lead to more effective clinical interventions.

Malaria in the 'Omics Era'.

Genomics has revolutionised the study of the biology of parasitic diseases. The first Eukaryotic parasite to have its genome sequenced was the malaria parasite Plasmodium falciparum. Since then, Plasmodium genomics has continued to lead the way in the study of the genome biology of parasites, both in breadth—the number of Plasmodium species’ genomes sequenced—and in depth—massive-scale genome re-sequencing of several key species. Here, we review some of the insights into the biology, evolution and population genetics of Plasmodium gained from genome sequencing, and look at potential new avenues in the future genome-scale study of its biology.

Plasmodium falciparum goes bananas for sex

The sexual blood stages of the human malaria parasite Plasmodium falciparum undergo a remarkable transformation from a roughly spherical shape to an elongated crescent or “falciform” morphology from which the species gets its name. In this review, the molecular events that drive this spectacular shape change are discussed and some questions that remain regarding the mechanistic underpinnings are posed. We speculate on the role of the shape changes in promoting sequestration and release of the developing gametocyte, thereby facilitating parasite survival in the host and underpinning transmission to the mosquito vector.

Erythrocyte CD55 mediates the internalization of Plasmodium falciparum parasites.

Invasion of human erythrocytes by the malaria parasite Plasmodium falciparum is a multi-step process. Previously, a forward genetic screen for P. falciparum host factors identified erythrocyte CD55 as essential for invasion, but its specific role and how it interfaces with the other factors that mediate this complex process are unknown. Using CRISPR-Cas9 editing, antibody-based inhibition, and live cell imaging, here we show that CD55 is specifically required for parasite internalization. Pre-invasion kinetics, erythrocyte deformability, and echinocytosis were not influenced by CD55, but entry was inhibited when CD55 was blocked or absent. Visualization of parasites attached to CD55-null erythrocytes points to a role for CD55 in stability and/or progression of the moving junction. Our findings demonstrate that CD55 acts after discharge of the parasite's rhoptry organelles, and plays a unique role relative to all other invasion receptors. As the requirement for CD55 is strain-transcendent, these results suggest that CD55 or its interacting partners may hold potential as therapeutic targets for malaria.

Identification of novel inner membrane complex and apical annuli proteins of the malaria parasite Plasmodium falciparum.

The inner membrane complex (IMC) is a defining feature of apicomplexan parasites, which confers stability and shape to the cell, functions as a scaffolding compartment during the formation of daughter cells and plays an important role in motility and invasion during different life cycle stages of these single-celled organisms. To explore the IMC proteome of the malaria parasite Plasmodium falciparum we applied a proximity-dependent biotin identification (BioID)-based proteomics approach, using the established IMC marker protein Photosensitized INA-Labelled protein 1 (PhIL1) as bait in asexual blood-stage parasites. Subsequent mass spectrometry-based peptide identification revealed enrichment of 12 known IMC proteins and several uncharacterized candidate proteins. We validated nine of these previously uncharacterized proteins by endogenous GFP-tagging. Six of these represent new IMC proteins, while three proteins have a distinct apical localization that most likely represents structures described as apical annuli in Toxoplasma gondii. Additionally, various Kelch13 interacting candidates were identified, suggesting an association of the Kelch13 compartment and the IMC in schizont and merozoite stages. This work extends the number of validated IMC proteins in the malaria parasite and reveals for the first time the existence of apical annuli proteins in P. falciparum. Additionally, it provides evidence for a spatial association between the Kelch13 compartment and the IMC in late blood-stage parasites.

Characterisation of complexes formed by parasite proteins exported into the host cell compartment of Plasmodium falciparum infected red blood cells.

During its intraerythrocytic life cycle, the human malaria parasite Plasmodium falciparum supplements its nutritional requirements by scavenging substrates from the plasma through the new permeability pathways (NPPs) installed in the red blood cell (RBC) membrane. Parasite proteins of the RhopH complex: CLAG3, RhopH2, RhopH3, have been implicated in NPP activity. Here, we studied 13 exported proteins previously hypothesised to interact with RhopH2, to study their potential contribution to the function of NPPs. NPP activity assays revealed that the 13 proteins do not appear to be individually important for NPP function, as conditional knockdown of these proteins had no effect on sorbitol uptake. Intriguingly, reciprocal immunoprecipitation assays showed that five of the 13 proteins interact with all members of the RhopH complex, with PF3D7_1401200 showing the strongest association. Mass spectrometry‐based proteomics further identified new protein complexes; a cytoskeletal complex and a Maurer's clefts/J‐dot complex, which overall helps clarify protein–protein interactions within the infected RBC (iRBC) and is suggestive of the potential trafficking route of the RhopH complex itself to the RBC membrane.

Plasmodium falciparum SURFIN4.1 forms an intermediate complex with PTEX components and Pf113 during export to the red blood cell.

Plasmodium falciparum malaria parasites export several hundred proteins to the cytoplasm of infected red blood cells (RBCs) to modify the cell environment suitable for their growth. A Plasmodium translocon of exported proteins (PTEX) is necessary for both soluble and integral membrane proteins to cross the parasitophorous vacuole (PV) membrane surrounding the parasite inside the RBC. However, the molecular composition of the translocation complex for integral membrane proteins is not fully characterized, especially at the parasite plasma membrane. To examine the translocation complex, here we used mini-SURFIN4.1, consisting of a short N-terminal region, a transmembrane region, and a cytoplasmic region of an exported integral membrane protein SURFIN4.1. We found that mini-SURFIN4.1 forms a translocation intermediate complex with core PTEX components, EXP2, HSP101, and PTEX150. We also found that several proteins are exposed to the PV space, including Pf113, an uncharacterized PTEX-associated protein. We determined that Pf113 localizes in dense granules at the merozoite stage and on the parasite periphery after RBC invasion. Using an inducible translocon-clogged mini-SURFIN4.1, we found that a stable translocation intermediate complex forms at the parasite plasma membrane and contains EXP2 and a processed form of Pf113. These results suggest a potential role of Pf113 for the translocation step of mini-SURFIN4.1, providing further insights into the translocation mechanisms for parasite integral membrane proteins.

Synthesis of New Triazolopyrazine Antimalarial Compounds.

A radical approach to late-stage functionalization using photoredox and Diversinate™ chemistry on the Open Source Malaria (OSM) triazolopyrazine scaffold (Series 4) resulted in the synthesis of 12 new analogues, which were characterized by NMR, UV, and MS data analysis. The structures of four triazolopyrazines were confirmed by X-ray crystal structure analysis. Several minor and unexpected side products were generated during these studies, including two resulting from a possible disproportionation reaction. All compounds were tested for their ability to inhibit the growth of the malaria parasite Plasmodium falciparum (3D7 and Dd2 strains) and for cytotoxicity against a human embryonic kidney (HEK293) cell line. Moderate antimalarial activity was observed for some of the compounds, with IC50 values ranging from 0.3 to >20 µM; none of the compounds displayed any toxicity against HEK293 at 80 µM.

Has artemisinin resistance emerged in Africa?

Earlier this century, Plasmodium falciparum malaria parasites developed the ability to evade the action of artemisinin antimalarials. This delayed clearance phenotype, referred to as artemisinin resistance, is widespread in the Greater Mekong Subregion of southeast Asia, where it is associated with treatment failures for some artemisinin-based combination therapies (ACTs), the drugs of choice to treat P falciparum malaria.1 However, there is little evidence of artemisinin resistance beyond southeast Asia.

Development of a bispecific immune engager using a recombinant malaria protein

As an immune evasion and survival strategy, the Plasmodium falciparum malaria parasite has evolved a protein named VAR2CSA. This protein mediates sequestration of infected red blood cells in the placenta through the interaction with a unique carbohydrate abundantly and exclusively present in the placenta. Cancer cells were found to share the same expression of this distinct carbohydrate, termed oncofetal chondroitin sulfate on their surface. In this study we have used a protein conjugation system to produce a bispecific immune engager, V-aCD3, based on recombinant VAR2CSA as the cancer targeting moiety and an anti-CD3 single-chain variable fragment linked to a single-chain Fc as the immune engager. Conjugation of these two proteins resulted in a single functional moiety that induced immune mediated killing of a broad range of cancer cells in vitro and facilitated tumor arrest in an orthotopic bladder cancer xenograft model.

Chemical Biology Tools To Investigate Malaria Parasites

Parasitic diseases like malaria tropica have been shaping human evolution and history since the beginning of mankind. After infection, the response of the human host ranges from asymptomatic to severe and may culminate in death. Therefore, proper examination of the parasite's biology is pivotal to deciphering unique molecular, biochemical and cell biological processes, which in turn ensure the identification of treatment strategies, such as potent drug targets and vaccine candidates. However, implementing molecular biology methods for genetic manipulation proves to be difficult for many parasite model organisms. The development of fast and straightforward applicable alternatives, for instance small‐molecule probes from the field of chemical biology, is essential. In this review, we will recapitulate the highlights of previous molecular and chemical biology approaches that have already created insight and understanding of the malaria parasite Plasmodium falciparum. We discuss current developments from the field of chemical biology and explore how their application could advance research into this parasite in the future. We anticipate that the described approaches will help to close knowledge gaps in the biology of P. falciparum and we hope that researchers will be inspired to use these methods to gain knowledge – with the aim of ending this devastating disease.

Evaluation of ethnopharmacologically selected Vitex negundo L. for In vitro antimalarial activity and secondary metabolite profiling

Ethanopharmacological relevanceLimited drugs, rise in drug resistance against frontline anti-malarial drugs, non-availability of efficacious vaccines and high cost of drug development hinders malaria intervention programs. Search for safe, effective and affordable plant based anti-malarial agents, thus becomes crucial and vital in the current scenario. The Vitex negundo L. is medicinal plant possessing a variety of pharmaceutically important compounds. The plant is used traditionally worldwide for the treatment of malaria including India and Malaysia by the indigenous tribes. In vitro studies have reported the anti-malarial use of the plant in traditional medicinal systems. Aim of the studyThe aim of the current study is to evaluate the traditionally used medicinal plants for in vitro anti-malarial activity against human malaria parasite Plasmodium falciparum and profiling secondary metabolite using spectroscopic and chromatographic methods. Chemical profiling of active secondary metabolites in the extracts was undertaken using LC-MS. Materials and methodsBased on the ethno-botanical data V. negundo L. was selected for in vitro anti-malarial activity against P. falciparum chloroquine-sensitive (3D7) and multidrug resistant (K1) strains using SYBR Green-I based fluorescence assay. Cytotoxicity of extracts was evaluated in VERO cell line using the MTT assay. Haemolysis assay was performed using human red blood cells. Secondary metabolites profiling was undertaken using chromatographic and spectroscopic analysis. Liquid chromatography analysis was performed using a C18, 150 X 2.1, 2.6 μm column with gradient mobile phase Solvent A: 95% (H2O: ACN), Solvent B: Acetonitrile, Solvent C: Methanol, Solvent D: 5 mM NH4 in 95:5 (H2O: ACN) at a constant flow rate of 0.250 ml/min. The LC-MS spectra were acquired in both positive and negative ion modes with electrospray ionization (ESI) source. ResultsThe anti-malarial active extract of V. negundo L. leaf exhibited potent anti-malarial activity with IC50 values of 7.21 μg/ml and 7.43 μg/ml against 3D7 and K1 strains, respectively with no evidence of significant cytotoxicity against mammalian cell line (VERO) and no toxicity as observed in haemolysis assay. The HPLC-LC-MS analysis of the extract led to identification of 73 compounds. We report for the first time the presence of Sabinene hydrate acetate, 5-Hydroxyoxindole, 2(3,4-dimethoxyphenyl)-6, 7-dimethoxychromen-4-one, Cyclotetracosa-1, 13-diene and 5, 7-Dimethoxyflavanone in the anti-malarial active extract of V. negundo L. leaf. Agnuside, Behenic acid and Globulol are some of the novel compounds with no reports of anti-malarial activity so far and require further evaluation in pure form for the development of potent anti-malarial compounds. ConclusionsThe result report and scientifically validate the traditional use of V. negundo L. for the treatment of malaria providing new avenues for anti-malarial drug development. Several novel and unknown compounds were identified that need to be further characterized for anti-malarial potential.

Targeting Gametocytes of the Malaria Parasite Plasmodium falciparum in a Functional Genomics Era: Next Steps.

Mosquito transmission of the deadly malaria parasite Plasmodium falciparum is mediated by mature sexual forms (gametocytes). Circulating in the vertebrate host, relatively few intraerythrocytic gametocytes are picked up during a bloodmeal to continue sexual development in the mosquito vector. Human-to-vector transmission thus represents an infection bottleneck in the parasite’s life cycle for therapeutic interventions to prevent malaria. Even though recent progress has been made in the identification of genetic factors linked to gametocytogenesis, a plethora of genes essential for sexual-stage development are yet to be unraveled. In this review, we revisit P. falciparum transmission biology by discussing targetable features of gametocytes and provide a perspective on a forward-genetic approach for identification of novel transmission-blocking candidates in the future.

The catalytic subunit of Plasmodium falciparum casein kinase 2 is essential for gametocytogenesis

Casein kinase 2 (CK2) is a pleiotropic kinase phosphorylating substrates in different cellular compartments in eukaryotes. In the malaria parasite Plasmodium falciparum, PfCK2 is vital for asexual proliferation of blood-stage parasites. Here, we applied CRISPR/Cas9-based gene editing to investigate the function of the PfCK2α catalytic subunit in gametocytes, the sexual forms of the parasite that are essential for malaria transmission. We show that PfCK2α localizes to the nucleus and cytoplasm in asexual and sexual parasites alike. Conditional knockdown of PfCK2α expression prevented the transition of stage IV into transmission-competent stage V gametocytes, whereas the conditional knockout of pfck2a completely blocked gametocyte maturation already at an earlier stage of sexual differentiation. In summary, our results demonstrate that PfCK2α is not only essential for asexual but also sexual development of P. falciparum blood-stage parasites and encourage studies exploring PfCK2α as a potential target for dual-active antimalarial drugs.