Development Of Plasmodium Falciparum Research Articles

A single cell atlas of sexual development in Plasmodium falciparum.

The developmental decision made by malaria parasites to become sexual underlies all malaria transmission. Here, we describe a rich atlas of short- and long-read single-cell transcriptomes of over 37,000 Plasmodium falciparum cells across intraerythrocytic asexual and sexual development. We used the atlas to explore transcriptional modules and exon usage along sexual development and expanded it to include malaria parasites collected from four Malian individuals naturally infected with multiple P. falciparum strains. We investigated genotypic and transcriptional heterogeneity within and among these wild strains at the single-cell level, finding differential expression between different strains even within the same host. These data are a key addition to the Malaria Cell Atlas interactive data resource, enabling a deeper understanding of the biology and diversity of transmission stages.

PfCAP-H is essential for assembly of condensin I complex and karyokinesis during asexual proliferation of Plasmodium falciparum.

Mitosis is a fundamental process for Plasmodium parasites, which plays a vital role in their survival within two distinct hosts-human and Anopheles mosquitoes. Despite its great significance, our comprehension of mitosis and its regulation remains limited. In eukaryotes, mitosis is regulated by one of the pivotal complexes known as condensin complexes. The condensin complexes are responsible for chromosome condensation, ensuring the faithful distribution of genetic material to daughter cells. While condensin complexes have recently been identified in Plasmodium spp., our understanding of how this complex is assembled and its precise functions during the blood stage development of Plasmodium falciparum remains largely unexplored. In this study, we investigate the role of a central protein, PfCAP-H, during the blood stage development of P. falciparum. Our findings reveal that PfCAP-H is essential and plays a pivotal role in upholding the structure of condensin I and facilitating karyokinesis.

Putative molecular markers of Plasmodium falciparum resistance to antimalarial drugs in malaria parasites from Ghana.

Antimalarial drugs including artemisinin-based combination therapy (ACT) regimens and sulphadoxine-pyrimethamine (SP) are used in Ghana for malaria therapeutics and prophylaxis respectively. The genetic basis of Plasmodium falciparum development of drug resistance involves single nucleotide polymorphisms in genes encoding proteins for multiple cellular and metabolic processes. The prevalence of single nucleotide polymorphisms in nine P. falciparum genes linked to ACT and SP resistance in the malaria parasite population was determined. Archived filter paper blood blot samples from patients aged 9 years and below with uncomplicated malaria reporting at 10 sentinel sites located in three ecological zones for the Malaria Therapeutic Efficacy Studies were used. The samples used were collected from 2007-2018 malaria transmission seasons and mutations in the genes were detected using PCR and Sanger sequencing. In all 1,142 samples were used for the study. For falcipain-2 gene (pffp2), Sanger sequencing was successful for 872 samples and were further analysed. The prevalence of the mutants was 45% (392/872) with pffp2 markers V51I and S59F occurring in 15.0% (128/872) and 3.0% (26/872) of the samples respectively. Prevalence of other P. falciparum gene mutations: coronin (pfcoronin) was 44.8% (37/90); cysteine desulfurase (pfnfs) was 73.9% (68/92); apicoplast ribosomal protein S10 (pfarps10) was 36.8% (35/95); ferredoxin (pffd) was 8.8% (8/91); multidrug resistance protein-1 (pfmrp1) was 95.2.0% (80/84); multidrug resistance protein-2 (pfmrp2) was 91.4% (32/35); dihydrofolate reductase (pfdhfr) was 99.0% (84/85); dihydropteroate synthase (pfdhps) was 72% (68/95). The observation of numerous mutations in these genes of interest in the Ghanaian isolates, some of which have been implicated in delayed parasite clearance is of great interest. The presence of these genotypes may account for the decline in the efficacies of ACT regimens being used to treat uncomplicated malaria in the country. The need for continuous monitoring of these genetic markers to give first-hand information on parasite susceptibility to antimalarial drugs to inform policy makers and stakeholders in malaria elimination in the country is further discussed.

Attractive targeted sugar bait: the pyrrole insecticide chlorfenapyr and the anti-malarial pharmaceutical artemether–lumefantrine arrest Plasmodium falciparum development inside wild pyrethroid-resistant Anopheles gambiae s.s. mosquitoes

BackgroundAttractive targeted sugar bait (ATSB) is a novel approach to vector control, offering an alternative mode of insecticide delivery via the insect alimentary canal, with potential to deliver a variety of compounds new to medical entomology and malaria control. Its potential to control mosquitoes was recently demonstrated in major field trials in Africa. The pyrrole chlorfenapyr is an insecticide new to malaria vector control, and through its unique mode of action—disruption of ATP mediated energy transfer in mitochondria—it may have direct action on energy transfer in the flight muscle cells of mosquitoes. It may also have potential to disrupt mitochondrial function in malarial parasites co-existing within the infected mosquito. However, little is known about the impact of such compounds on vector competence in mosquitoes responsible for malaria transmission.MethodsIn this study, ATSBs containing chlorfenapyr insecticide and, as a positive control, the anti-malarial drugs artemether/lumefantrine (A/L) were compared for their effect on Plasmodium falciparum development in wild pyrethroid-resistant Anopheles gambiae sensu stricto (s.s.) and for their capacity to reduce vector competence. Female mosquitoes were exposed to ATSB containing either sublethal dose of chlorfenapyr (CFP: 0.025%) or concentrations of A/L ranging from 0.4/2.4 mg/ml to 2.4/14.4 mg/ml, either shortly before or after taking infective blood meals. The impact of their component compounds on the prevalence and intensity of P. falciparum infection were compared between treatments.ResultsBoth the prevalence and intensity of infection were significantly reduced in mosquitoes exposed to either A/L or chlorfenapyr, compared to unexposed negative control mosquitoes. The A/L dose (2.4/14.4 mg/ml) totally erased P. falciparum parasites: 0% prevalence of infection in female mosquitoes exposed compared to 62% of infection in negative controls (df = 1, χ2 = 31.23 p < 0.001). The dose of chlorfenapyr (0.025%) that killed < 20% females in ATSB showed a reduction in oocyte density of 95% per midgut (0.18/3.43 per midgut).ConclusionThese results are evidence that chlorfenapyr, in addition to its direct killing effect on the vector, has the capacity to block Plasmodium transmission by interfering with oocyte development inside pyrethroid-resistant mosquitoes, and through this dual action may potentiate its impact under field conditions.

Plasmodium falciparum Development from Gametocyte to Oocyst: Insight from Functional Studies.

Malaria elimination may never succeed without the implementation of transmission-blocking strategies. The transmission of Plasmodium spp. parasites from the human host to the mosquito vector depends on circulating gametocytes in the peripheral blood of the vertebrate host. Once ingested by the mosquito during blood meals, these sexual forms undergo a series of radical morphological and metabolic changes to survive and progress from the gut to the salivary glands, where they will be waiting to be injected into the vertebrate host. The design of effective transmission-blocking strategies requires a thorough understanding of all the mechanisms that drive the development of gametocytes, gametes, sexual reproduction, and subsequent differentiation within the mosquito. The drastic changes in Plasmodium falciparum shape and function throughout its life cycle rely on the tight regulation of stage-specific gene expression. This review outlines the mechanisms involved in Plasmodium falciparum sexual stage development in both the human and mosquito vector, and zygote to oocyst differentiation. Functional studies unravel mechanisms employed by P. falciparum to orchestrate the expression of stage-specific functional products required to succeed in its complex life cycle, thus providing us with potential targets for developing new therapeutics. These mechanisms are based on studies conducted with various Plasmodium species, including predominantly P. falciparum and the rodent malaria parasites P. berghei. However, the great potential of epigenetics, genomics, transcriptomics, proteomics, and functional genetic studies to improve the understanding of malaria as a disease remains partly untapped because of limitations in studies using human malaria parasites and field isolates.

Phenotypic Screens Identify Genetic Factors Associated with Gametocyte Development in the Human Malaria Parasite Plasmodium falciparum.

Transmission of the deadly malaria parasite Plasmodium falciparum from humans to mosquitoes is achieved by specialized intraerythrocytic sexual forms called gametocytes. Though the crucial regulatory mechanisms leading to gametocyte commitment have recently come to light, networks of genes that control sexual development remain to be elucidated. Here, we report a pooled-mutant screen to identify genes associated with gametocyte development in P. falciparum. Our results categorized genes that modulate gametocyte progression as hypoproducers or hyperproducers of gametocytes, and the in-depth analysis of individual clones confirmed phenotypes in sexual commitment rates and putative functions in gametocyte development. We present a new set of genes that have not been implicated in gametocytogenesis before and demonstrate the potential of forward genetic screens in isolating genes impacting parasite sexual biology, an exciting step toward the discovery of new antimalarials for a globally significant pathogen. IMPORTANCE Blocking human-to-vector transmission is an essential step toward malaria elimination. Gametocytes are solely responsible for achieving this transmission and represent an opportunity for therapeutic intervention. While these falciform-shaped parasite stages were first discovered in the 1880s, our understanding of the genetic determinants responsible for their formation and molecular mechanisms that drive their development is limited. In this work, we developed a scalable screening methodology with piggyBac mutants to identify genes that influence the development of gametocytes in the most lethal human malaria parasite, P. falciparum. By doing so, we lay the foundation for large-scale functional genomic studies specifically designed to address remaining questions about sexual commitment, maturation, and mosquito infection in P. falciparum. Such functional genetic screens will serve to expedite the identification of essential pathways and processes for the development of novel transmission-blocking agents.

Single-Cell Transcriptomics To Define Plasmodium falciparum Stage Transition in the Mosquito Midgut.

Malaria inflicts the highest rate of morbidity and mortality among the vector-borne diseases. The dramatic bottleneck of parasite numbers that occurs in the gut of the obligatory mosquito vector provides a promising target for novel control strategies. Using single-cell transcriptomics, we analyzed Plasmodium falciparum development in the mosquito gut, from unfertilized female gametes through the first 20 h after blood feeding, including the zygote and ookinete stages. This study revealed the temporal gene expression of the ApiAP2 family of transcription factors and of parasite stress genes in response to the harsh environment of the mosquito midgut. Further, employing structural protein prediction analyses, we found several upregulated genes predicted to encode intrinsically disordered proteins (IDPs), a category of proteins known for their importance in regulation of transcription, translation, and protein-protein interactions. IDPs are known for their antigenic properties and may serve as suitable targets for antibody- or peptide-based transmission suppression strategies. In total, this study uncovers the P. falciparum transcriptome from early to late parasite development in the mosquito midgut, inside its natural vector, which provides an important resource for future malaria transmission-blocking initiatives. IMPORTANCE The malaria parasite Plasmodium falciparum causes more than half a million deaths per year. The current treatment regimen targets the symptom-causing blood stage inside the human host. However, recent incentives in the field call for novel interventions to block parasite transmission from humans to the mosquito vector. Therefore, we need to better understand the parasite biology during its development inside the mosquito, including a deeper understanding of the expression of genes controlling parasite progression during these stages. Here, we have generated single-cell transcriptome data, covering P. falciparum's development, from gamete to ookinete inside the mosquito midgut, uncovering previously untapped parasite biology, including a repertoire of novel biomarkers to be explored in future transmission-blocking efforts. We anticipate that our study provides an important resource, which can be further explored to improve our understanding of the parasite biology as well as aid in guiding future malaria intervention strategies.

Physiological jump in erythrocyte redox potential during Plasmodium falciparum development occurs independent of the sickle cell trait

The redox state of the host-parasite unit has been hypothesized to play a central role for the fitness of the intraerythrocytic blood stages of the human malaria parasite Plasmodium falciparum. In particular, hemoglobinopathies have been suggested to cause a more oxidizing environment, thereby protecting from severe malaria. Here we determined the redox potential of infected wild-type (hemoglobin AA) or sickle trait (hemoglobin AS) erythrocytes using parasite-encoded variants of the redox-sensitive green-fluorescent protein 2 (roGFP2). Our non-invasive roGFP2 single-cell measurements revealed a reducing steady-state redox potential of −304 ± 11 mV for the erythrocyte cytosol during ring-stage development and a rather sudden oxidation to −278 ± 12 mV during trophozoite-stage development around 28 h post invasion. There was no significant difference between wild-type or sickle trait erythrocytes regarding the stage dependence and the detected increase of the redox potential during the intraerythrocytic life cycle. The steady-state redox potential of the parasite cytosol, between −304 and −313 mV, was highly reducing throughout the life cycle. The redox potential in the parasitophorous vacuole at the interface between the secretory pathway and the erythrocyte was −284 ± 10 mV and remained stable during trophozoite-stage development with implications for the export of disulfide-containing proteins. In summary, P. falciparum blood stage development from the late ring to the early trophozoite stage causes a physiological jump in erythrocyte redox potential irrespective of the presence or absence of hemoglobin S.

Chemically induced phenotypes during the blood stage development of Plasmodium falciparum as indicators of the drug mode of action

Malaria remains a health and economic burden, particularly in marginalized populations worldwide. The current strategies for combating malaria rely on eliminating the mosquito vector, using insecticide-treated nets, and other management policies or through the administration of small molecule drugs to perturb the intra-erythrocytic development of the parasite. However, resistance against commonly used drugs such as artemisinin has recently become a concern necessitating the identification of novel pharmacophores with unique mechanisms of action. This review summarizes the various life-stage events of the malaria parasite, Plasmodium falciparum, during the in vitro development, which can be targeted by different classes of small molecules. We also describe various chemically induced phenotypes and methods to ascertain and validate drug-induced changes to derive early insights into which cellular mechanisms are affected.

Vaccination with a structure-based stabilized version of malarial antigen Pfs48/45 elicits ultra-potent transmission-blocking antibody responses

SummaryMalaria transmission-blocking vaccines (TBVs) aim to elicit human antibodies that inhibit sporogonic development of Plasmodium falciparum in mosquitoes, thereby preventing onward transmission. Pfs48/45 is a leading clinical TBV candidate antigen and is recognized by the most potent transmission-blocking monoclonal antibody (mAb) yet described; still, clinical development of Pfs48/45 antigens has been hindered, largely by its poor biochemical characteristics. Here, we used structure-based computational approaches to design Pfs48/45 antigens stabilized in the conformation recognized by the most potently inhibitory mAb, achieving >25°C higher thermostability compared with the wild-type protein. Antibodies elicited in mice immunized with these engineered antigens displayed on liposome-based or protein nanoparticle-based vaccine platforms exhibited 1–2 orders of magnitude superior transmission-reducing activity, compared with immunogens bearing the wild-type antigen, driven by improved antibody quality. Our data provide the founding principles for using molecular stabilization solely from antibody structure-function information to drive improved immune responses against a parasitic vaccine target.

Refolding and characterization of a diabody against Pfs25, a vaccine candidate of Plasmodium falciparum

Pfs25, a vaccine candidate, expressed on the surface of the malarial parasite, plays an important role in the development of Plasmodium falciparum. 1269, a monoclonal antibody targeting the epidermal growth factor-like domain 1 and epidermal growth factor-like domain 3 of Pfs25, blocks the transmission of parasites in mosquitoes. In this study, we refolded 1269-Db, a dimeric antibody fragment referred as diabody, designed from 1269, with a yield of 3 mg/litre of bacterial culture. Structural integrity of the protein was validated with thermal stability, disulphide bond analysis and glutaraldehyde crosslinking experiments. To evaluate the functionality of 1269-Db, recombinant monomeric MBP-Pfs25 was produced from bacteria. Qualitative binding assays demonstrated that 1269-Db recognized the epitopes on Pfs25 in its native, but not the denatured state. An apparent KD of 2.6 nM was determined for 1269-Db with monomeric MBP-Pfs25, using isothermal titration calorimetry. 1269-Db recognized the periphery of zygotes/ookinetes, demonstrating recognition of Pfs25, expressed on the surface of the parasite. As the established refolding method resulted in a functional diabody, the optimized method pipeline for 1269-Db can potentially facilitate engineering of antibody fragments with desired properties.

The Knock-Down of the Chloroquine Resistance Transporter PfCRT Is Linked to Oligopeptide Handling in Plasmodium falciparum.

ABSTRACTThe chloroquine resistance transporter, PfCRT, is an essential factor during intraerythrocytic development of the human malaria parasite Plasmodium falciparum. PfCRT resides at the digestive vacuole of the parasite, where hemoglobin taken up by the parasite from its host cell is degraded. PfCRT can acquire several mutations that render PfCRT a drug transporting system expelling compounds targeting hemoglobin degradation from the digestive vacuole. The non-drug related function of PfCRT is less clear, although a recent study has suggested a role in oligopeptide transport based on studies conducted in a heterologous expression system. The uncertainty about the natural function of PfCRT is partly due to a lack of a null mutant and a dearth of functional assays in the parasite. Here, we report on the generation of a conditional PfCRT knock-down mutant in P. falciparum. The mutant accumulated oligopeptides 2 to at least 8 residues in length under knock-down conditions, as shown by comparative global metabolomics. The accumulated oligopeptides were structurally diverse, had an isoelectric point between 4.0 and 5.4 and were electrically neutral or carried a single charge at the digestive vacuolar pH of 5.2. Fluorescently labeled dipeptides and live cell imaging identified the digestive vacuole as the compartment where oligopeptides accumulated. Our findings suggest a function of PfCRT in oligopeptide transport across the digestive vacuolar membrane in P. falciparum and associated with it a role in nutrient acquisition and the maintenance of the colloid osmotic balance.IMPORTANCE The chloroquine resistance transporter, PfCRT, is important for the survival of the human malaria parasite Plasmodium falciparum. It increases the tolerance to many antimalarial drugs, and it is essential for the development of the parasite within red blood cells. While we understand the role of PfCRT in drug resistance in ever increasing detail, the non-drug resistance functions are still debated. Identifying the natural substrate of PfCRT has been hampered by a paucity of functional assays to test putative substrates in the parasite system and the absence of a parasite mutant deficient for the PfCRT encoding gene. By generating a conditional PfCRT knock-down mutant, together with comparative metabolomics and uptake studies using fluorescently labeled oligopeptides, we could show that PfCRT is an oligopeptide transporter. The oligopeptides were structurally diverse and were electrically neutral or carried a single charge. Our data support a function of PfCRT in oligopeptide transport.

Heterologous Expression and Evaluation of Novel Plasmodium falciparum Transmission Blocking Vaccine Candidates.

Malaria transmission blocking vaccines (TBV) aim to induce antibodies that can interrupt Plasmodium falciparum development in the mosquito midgut and thereby prevent onward malaria transmission. A limited number of TBV candidates have been identified and only three (Pfs25, Pfs230 and Pfs48/45) have entered clinical testing. While one of these candidates may emerge as a highly potent TBV candidate, it is premature to determine if they will generate sufficiently potent and sustained responses. It is therefore important to explore novel candidate antigens. We recently analyzed sera from naturally exposed individuals and found that the presence and/or intensity of antibodies against 12 novel putative surface expressed gametocyte antigens was associated with transmission reducing activity. In this study, protein fragments of these novel TBV candidates were designed and heterologously expressed in Drosophila melanogaster S2 cells and Lactococcus lactis. Eleven protein fragments, covering seven TBV candidates, were successfully produced. All tested antigens were recognized by antibodies from individuals living in malaria-endemic areas, indicating that native epitopes are present. All antigens induced antigen-specific antibody responses in mice. Two antigens induced antibodies that recognized a native protein in gametocyte extract, and antibodies elicited by four antigens recognized whole gametocytes. In particular, we found that antigen Pf3D7_0305300, a putative transporter, is abundantly expressed on the surface of gametocytes. However, none of the seven novel TBV candidates expressed here induced an antibody response that reduced parasite development in the mosquito midgut as assessed in the standard membrane feeding assay. Altogether, the antigen fragments used in this study did not prove to be promising transmission blocking vaccine constructs, but led to the identification of two gametocyte surface proteins that may provide new leads for studying gametocyte biology.

Assay Development and Identification of the First Plasmodium falciparum 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase Inhibitors.

In the fight towards eradication of malaria, identifying compounds active against new drug targets constitutes a key approach. Plasmodium falciparum 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (PfHPPK) has been advanced as a promising target, as being part of the parasite essential folate biosynthesis pathway while having no orthologue in the human genome. However, no drug discovery efforts have been reported on this enzyme. In this study, we conducted a three-step screening of our in-house antifolate library against PfHPPK using a newly designed PfHPPK-GFP protein construct. Combining virtual screening, differential scanning fluorimetry and enzymatic assay, we identified 14 compounds active against PfHPPK. Compounds’ binding modes were investigated by molecular docking, suggesting competitive binding with the HMDP substrate. Cytotoxicity and in vitro ADME properties of hit compounds were also assessed, showing good metabolic stability and low toxicity. The most active compounds displayed low micromolar IC50 against drug-resistant parasites. The reported hit compounds constitute a good starting point for inhibitor development against PfHPPK, as an alternative approach to tackle the malaria parasite.

An In Silico Study of the Interactions of Alkaloids from Cryptolepis sanguinolenta with Plasmodium falciparum Dihydrofolate Reductase and Dihydroorotate Dehydrogenase

The Plasmodium falciparum dihydrofolate reductase (PfDHFR) and dihydroorotate dehydrogenase (PfDHODH) are essential for Plasmodium falciparum growth and development, and have been validated as targets for the development of new antimalarial agents. Several alkaloids isolated from Cryptolepis sanguinolenta have been reported to have antiplasmodial activity, but their protein targets are unknown. Therefore, molecular docking and molecular dynamics simulations were used to investigate the interactions and stability of the alkaloids with PfDHFR and PfDHODH. Based on physicochemical characteristics, alkaloids were grouped as sterically bulky (sb) or planar (pg). Docking results revealed strong binding affinities (−6.0 to −13.4 kcal/mol) of the alkaloids against PfDHODH and various strains of PfDHFR while interacting with key residues such as Asp54 and Phe58 in PfDHFR. The pg alkaloids had high binding affinity and preference for the inhibitor binding domain over the flavin mononucleotide (FMN) binding domain in PfDHODH due to size considerations. From the molecular dynamics trajectories, protein-alkaloid complexes were stable throughout the simulation, with supporting evidence from root mean square deviations, root mean square fluctuations, radius of gyration, free binding energies, and other parameters. We report herein that biscryptolepine and cryptomisrine (sb class), as well as cryptolepinone, cryptoheptine, cryptolepine, and neocryptolepine (pg class), are capable of inhibiting PfDHFR effectively in pyrimethamine sensitive and resistant cells. Also, our results show that alkaloids of the pg class can inhibit PfDHODH as FMN decoys, as well as direct enzyme inhibitors, thereby halting crucial protein function.

Cover Feature: The Catalytic Mechanism of Pdx2 Glutaminase Driven by a Cys‐His‐Glu Triad: A Computational Study (ChemBioChem 9/2022)

An atomistic description of Pdx2 catalytic mechanism is provided by QM/MM methodologies. Pdx2, the glutaminase subunit of pyridoxal 5’-phosphate (PLP) synthase, is characterized by a Cys-His-Glu catalytic triad used to deaminate glutamine to glutamate. While being absent in humans, Pdx2 is essential for the development and replication of the protozoan Plasmodium falciparum and Mycobacterium tuberculosis pathogens, which are responsible for two deadly diseases, malaria and tuberculosis, respectively. Pdx2 is thus a promising drug target for fighting these diseases. More information can be found in the Full Paper by A. F. Pina et al.

Synthesis and antiplasmodial activity of regioisomers and epimers of second-generation dual acting ivermectin hybrids

With its strong effect on vector-borne diseases, and insecticidal effect on mosquito vectors of malaria, inhibition of sporogonic and blood-stage development of Plasmodium falciparum, as well as in vitro and in vivo impairment of the P. berghei development inside hepatocytes, ivermectin (IVM) continues to represent an antimalarial therapeutic worthy of investigation. The in vitro activity of the first-generation IVM hybrids synthesized by appending the IVM macrolide with heterocyclic and organometallic antimalarial pharmacophores, against the blood-stage and liver-stage infections by Plasmodium parasites prompted us to design second-generation molecular hybrids of IVM. Here, a structural modification of IVM to produce novel molecular hybrids by using sub-structures of 4- and 8-aminoquinolines, the time-tested antiplasmodial agents used for treating the blood and hepatic stage of Plasmodium infections, respectively, is presented. Successful isolation of regioisomers and epimers has been demonstrated, and the evaluation of their in vitro antiplasmodial activity against both the blood stages of P. falciparum and the hepatic stages of P. berghei have been undertaken. These compounds displayed structure-dependent antiplasmodial activity, in the nM range, which was more potent than that of IVM, its aglycon or primaquine, highlighting the superiority of this hybridization strategy in designing new antiplasmodial agents.

Plasmodium Resistance to Artemisinin Derivates due to Kelch-3 Gene Mutation

Artemisinin class of antimalarial drugs play an important role in controlling falciparum malaria after the emergence of resistance of Plasmodium falciparum to other antimalarial drugs such as chloroquine, sulfadoxine-pyrimethamine and mefloquine. Therefore, the presence of Plasmodium falciparum resistance to this class of drugs is threat to global efforts to eliminate this disease. Resistance of Plasmodium falciparum to artemisinin recently known to be associated with mutations in the propeller domain of the kelch-13 (K13) Plasmodium falciparum gene. The incidence of Plasmodium falciparum resistance due to mutations in the K13 gene, among others, can be found in Cambodia, Laos, Vietnam, China, Myanmar, Thailand and Africa. The presence of mutations in this gene will change the response of Plasmodium falciparum against oxidative stress induced by artemisinin by involving the proteasome-ubiquitin pathway. In addition, mutation K13 will also change the levels of PI3K and PI3P in the body of Plasmodium falciparum. PI3K and PI3P are lipids that essential for the development of Plasmodium falciparum from ring stage to schizont. Resistance to artemisinin will also provide phenotypic changes in the life cycle of Plasmodium falciparum in the form of elongation at the stage ring and transient shortening in trophozoite development. This resistance incident can be overcome, among others by prolonging the duration of treatment (from a 3-day regimen to a 4-day regimen) and combining artemisinin with proteasome inhibitors.

Specific sub fractions from Terminalia mantaly (H. Perrier) extracts potently inhibit Plasmodium falciparum rings, merozoite egress and invasion

Ethnopharmacological relevanceTerminalia mantaly (H. Perrier) and Terminalia superba (Engl. & Diels) are sources of treatment for various diseases, including malaria and/or related symptoms in parts of Southwestern Cameroon. However, there is limited information on the extent of the antiplasmodial potential of their extracts. Aim of the studyThe present study was designed to investigate the antiplasmodial potential of chromatographic sub fractions (SFs) from promising fractions of Terminalia mantaly (Tm) [TmsbwChl, the chloroform fraction from water extract of Tm, IC50 (μg/mL) PfINDO: 0.56, Pf3D7: 1.12; SI > 357 (HEK/PfINDO) & 178 (HEK/Pf3D7)] and Terminalia superba (Ts) [TsrmEA, the ethyl acetate fraction from methanolic extract of Ts, IC50 (μg/mL) PfINDO: 1.82, Pf3D7: 1.65; SI > 109 (HEK/PfINDO) & 121 (HEK/Pf3D7)] obtained from previous studies. The SFs were tested against Plasmodium falciparum 3D7 (Pf3D7-chloroquine sensitive) and INDO (PfINDO-chloroquine resistant) strains in culture. Also, the phytochemical profile of potent SFs was determined and finally, the inhibition of the asexual blood stages of Plasmodium falciparum by the SFs with the highest promise was assessed. Material and methodsSelected SFs were submitted to a second bio-guided fractionation using silica gel column chromatography. The partial phytochemical composition of potent antiplasmodial SFs was determined using gas chromatography coupled to mass spectrometry (GC–MS). The SYBR Green I-based fluorescence microtiter plate assay was used to monitor the growth of Plasmodium falciparum parasites in culture in the presence or absence of extracts. Microscopy and flow cytometry counting was used to assess the Plasmodium falciparum stage–specific inhibition and post–drug exposure growth suppression by highly potent extracts. ResultsTwenty-one of the 39 SFs afforded from TmsbwChl showed activity (IC50: 0.29–4.74 μg/mL) against both Pf3D7 and PfINDO strains. Of note, eight SFs namely, Tm25, Tm28-30, Tm34-36 and Tm38, exerted highly potent antiplasmodial activity (IC50 < 1 μg/mL) with IC50PfINDO: 0.41–0.84 μg/mL and IC50Pf3D7: 0.29–0.68 μg/mL. They also displayed very high selectivity (50 < SIPfINDO, SIPf3D7 > 344) on the two Plasmodial strains. On the other hand, 7 SFs (SFs Ts03, Ts04, Ts06, Ts09, Ts10, Ts12 and Ts13) from TsrmEA showed promising inhibitory potential against both parasite strains (IC50: 2.01–5.14 μg/mL). Sub fraction Tm36 (IC50PfINDO: 0.41 μg/mL, SIPfINDO > 243; IC50Pf3D7: 0.29 μg/mL, SIPf3D7 > 344) showed the highest promise. The GC–MS analysis of the 8 selected SFs led to the identification of 99 phytometabolites, with D-limonene (2), benzaldehyde (12), carvone (13), caryophyllene (35), hexadecanoic acid, methyl ester (74) and 9-octadecenoic acid, methyl ester (82) being the main constituents. Sub fractions Tm28, Tm29, Tm30, Tm36 and Tm38 inhibited all the three intraerythrocytic stages of P. falciparum, with strong potency against ring stage development, merozoite egress and invasion processes. ConclusionsThis study has identified highly potent antiplasmodial SFs from Terminalia mantaly with significant activity on the intraerythrocytic development of Plasmodium falciparum. These SFs qualify as promising sources of novel antiplasmodial lead compounds. Further purification and characterization studies are expected to unravel molecular targets in rings and merozoites.

PI4-kinase and PfCDPK7 signaling regulate phospholipid biosynthesis in Plasmodium falciparum.

PfCDPK7 is an atypical member of the calcium-dependent protein kinase (CDPK) family and is crucial for the development of Plasmodium falciparum. However, the mechanisms whereby PfCDPK7 regulates parasite development remain unknown. Here, we perform quantitative phosphoproteomics and phospholipid analysis and find that PfCDPK7 promotes phosphatidylcholine (PC) synthesis by regulating two key enzymes involved in PC synthesis, phosphoethanolamine-N-methyltransferase (PMT) and ethanolamine kinase (EK). In the absence of PfCDPK7, both enzymes are hypophosphorylated and PMT is degraded. We further find that PfCDPK7 interacts with 4'-phosphorylated phosphoinositides (PIPs) generated by PI4-kinase. Inhibition of PI4K activity disrupts the vesicular localization PfCDPK7. P.falciparum PI4-kinase, PfPI4K is a prominent drug target and one of its inhibitors, MMV39048, has reached Phase I clinical trials. Using this inhibitor, we demonstrate that PfPI4K controls phospholipid biosynthesis and may act in part by regulating PfCDPK7 localization and activity. These studies not only unravel a signaling pathway involving PfPI4K/4'-PIPs and PfCDPK7 but also provide novel insights into the mechanism of action of a promising series of candidate anti-malarial drugs.