Development Of Plasmodium Falciparum Research Articles

A Plasmodium falciparum MORC protein complex modulates epigenetic control of gene expression through interaction with heterochromatin

Dynamic control of gene expression is critical for blood stage development of malaria parasites. Here, we used multi-omic analyses to investigate transcriptional regulation by the chromatin-associated microrchidia protein, MORC, during asexual blood stage development of the human malaria parasite Plasmodium falciparum. We show that PfMORC (PF3D7_1468100) interacts with a suite of nuclear proteins, including APETALA2 (ApiAP2) transcription factors (PfAP2-G5, PfAP2-O5, PfAP2-I, PF3D7_0420300, PF3D7_0613800, PF3D7_1107800, and PF3D7_1239200), a DNA helicase DS60 (PF3D7_1227100), and other chromatin remodelers (PfCHD1 and PfEELM2). Transcriptomic analysis of PfMORCHA-glmS knockdown parasites revealed 163 differentially expressed genes belonging to hypervariable multigene families, along with upregulation of genes mostly involved in host cell invasion. In vivo genome-wide chromatin occupancy analysis during both trophozoite and schizont stages of development demonstrates that PfMORC is recruited to repressed, multigene families, including the var genes in subtelomeric chromosomal regions. Collectively, we find that PfMORC is found in chromatin complexes that play a role in the epigenetic control of asexual blood stage transcriptional regulation and chromatin organization.

A Plasmodium falciparum MORC protein complex modulates epigenetic control of gene expression through interaction with heterochromatin.

Dynamic control of gene expression is critical for blood stage development of malaria parasites. Here, we used multi-omic analyses to investigate transcriptional regulation by the chromatin-associated microrchidia protein, MORC, during asexual blood stage development of the human malaria parasite Plasmodium falciparum. We show that PfMORC (PF3D7_1468100) interacts with a suite of nuclear proteins, including APETALA2 (ApiAP2) transcription factors (PfAP2-G5, PfAP2-O5, PfAP2-I, PF3D7_0420300, PF3D7_0613800, PF3D7_1107800, and PF3D7_1239200), a DNA helicase DS60 (PF3D7_1227100), and other chromatin remodelers (PfCHD1 and PfEELM2). Transcriptomic analysis of PfMORCHA-glmS knockdown parasites revealed 163 differentially expressed genes belonging to hypervariable multigene families, along with upregulation of genes mostly involved in host cell invasion. In vivo genome-wide chromatin occupancy analysis during both trophozoite and schizont stages of development demonstrates that PfMORC is recruited to repressed, multigene families, including the var genes in subtelomeric chromosomal regions. Collectively, we find that PfMORC is found in chromatin complexes that play a role in the epigenetic control of asexual blood stage transcriptional regulation and chromatin organization.

Heterochromatin dynamics during the initial stages of sexual development in Plasmodium falciparum

Asexual replication of Plasmodium falciparum in the human blood results in exponential parasite growth and causes all clinical symptoms of malaria. However, at each round of the replicative cycle, some parasites convert into sexual precursors called gametocytes, which develop through different stages until they become infective to mosquito vectors. The genome-wide distribution of heterochromatin, a type of chromatin generally refractory to gene expression, is identical at all asexual blood stages, but is altered in stage II/III and more mature gametocytes. However, it is not known if these changes occur concomitantly with sexual conversion or at a later time during gametocyte development. Using a transgenic line in which massive sexual conversion can be conditionally induced, we show that the genome-wide distribution of heterochromatin at the initial stages of sexual development (i.e., sexual rings and stage I gametocytes) is almost identical to asexual blood stages, and major changes do not occur until stage II/III. However, we found that at loci with heterochromatin alterations, transcriptional changes associated with sexual development typically precede, rather than follow, changes in heterochromatin occupancy.

Development of peptoid-based heteroaryl-decorated histone deacetylase (HDAC) inhibitors with dual-stage antiplasmodial activity

Dynamics of epigenetic modifications such as acetylation and deacetylation of histone proteins have been shown to be crucial for the life cycle development and survival of Plasmodium falciparum, the deadliest malaria parasite. In this study, we present a novel series of peptoid-based histone deacetylase (HDAC) inhibitors incorporating nitrogen-containing bicyclic heteroaryl residues as a new generation of antiplasmodial peptoid-based HDAC inhibitors. We synthesized the HDAC inhibitors by an efficient multicomponent protocol based on the Ugi four-component reaction. The subsequent screening of 16 compounds from our mini-library identified 6i as the most promising candidate, demonstrating potent activity against asexual blood-stage parasites (IC50Pf3D7 = 30 nM; IC50PfDd2 = 98 nM), low submicromolar activity against liver-stage parasites (IC50PbEEF = 0.25 μM), excellent microsomal stability (t1/2 > 60 min), and low cytotoxicity to HEK293 cells (IC50 = 136 μM).

Host cell CRISPR genomics and modelling reveal shared metabolic vulnerabilities in the intracellular development of Plasmodium falciparum and related hemoparasites

Parasitic diseases, particularly malaria (caused by Plasmodium falciparum) and theileriosis (caused by Theileria spp.), profoundly impact global health and the socioeconomic well-being of lower-income countries. Despite recent advances, identifying host metabolic proteins essential for these auxotrophic pathogens remains challenging. Here, we generate a novel metabolic model of human hepatocytes infected with P. falciparum and integrate it with a genome-wide CRISPR knockout screen targeting Theileria-infected cells to pinpoint shared vulnerabilities. We identify key host metabolic enzymes critical for the intracellular survival of both of these lethal hemoparasites. Remarkably, among the metabolic proteins identified by our synergistic approach, we find that host purine and heme biosynthetic enzymes are essential for the intracellular survival of P. falciparum and Theileria, while other host enzymes are only essential under certain metabolic conditions, highlighting P. falciparum’s adaptability and ability to scavenge nutrients selectively. Unexpectedly, host porphyrins emerge as being essential for both parasites. The shared vulnerabilities open new avenues for developing more effective therapies against these debilitating diseases, with the potential for broader applicability in combating apicomplexan infections.

Lipid droplet dynamics are essential for the development of the malaria parasite Plasmodium falciparum.

Lipid droplets (LDs) are organelles that are central to lipid and energy homeostasis across all eukaryotes. In the malaria-causing parasite Plasmodium falciparum the roles of LDs in lipid acquisition from its host cells and their metabolism are poorly understood, despite the high demand for lipids in parasite membrane synthesis. We systematically characterised LD size, composition and dynamics across the disease-causing blood infection. Applying split fluorescence emission analysis and three-dimensional (3D) focused ion beam-scanning electron microscopy (FIB-SEM), we observed a decrease in LD size in late schizont stages. LD contraction likely signifies a switch from lipid accumulation to lipid utilisation in preparation for parasite egress from host red blood cells. We demonstrate connections between LDs and several parasite organelles, pointing to potential functional interactions. Chemical inhibition of triacylglyerol (TAG) synthesis or breakdown revealed essential LD functions for schizogony and in counteracting lipid toxicity. The dynamics of lipid synthesis, storage and utilisation in P. falciparum LDs might provide a target for new anti-malarial intervention strategies.

Functionality of the V-type ATPase during asexual growth and development of Plasmodium falciparum

Vacuolar type ATPases (V-type ATPases) are highly conserved hetero-multisubunit proton pumping machineries found in all eukaryotes. They utilize ATP hydrolysis to pump protons, acidifying intracellular or extracellular compartments, and are thus crucial for various biological processes. Despite their evolutionary conservation in malaria parasites, this proton pump remains understudied. To understand the localization and biological functions of Plasmodium falciparum V-type ATPase, we employed CRISPR/Cas9 to endogenously tag the subunit A of the V1 domain. V1A (PF3D7_1311900) was tagged with a triple hemagglutinin epitope and the TetR-DOZI-aptamer system for conditional expression under the regulation of anhydrotetracycline. Via immunofluorescence assays, we identified that V-type ATPase is expressed throughout the intraerythrocytic developmental cycle and is mainly localized to the digestive vacuole and parasite plasma membrane. Immuno-electron microscopy further revealed that V-type ATPase is also localized on secretory organelles in merozoites. Knockdown of V1A led to cytosolic pH imbalance and blockage of hemoglobin digestion in the digestive vacuole, resulting in an arrest of parasite development in the trophozoite-stage and, ultimately, parasite demise. Using bafilomycin A1, a specific inhibitor of V-type ATPases, we found that the P.falciparum V-type ATPase is likely involved in parasite invasion but is not critical for ring-stage development. Further, we detected a large molecular weight complex in blue native-PAGE (∼1.0MDa), corresponding to the total molecular weights of V1 and Vo domains. Together, we show that V-type ATPase is localized to multiple subcellular compartments in P.falciparum, and its functionality throughout the asexual cycle varies depending on the parasite developmental stages.

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.

Combining transgenesis with paratransgenesis to fight malaria.

Malaria is among the deadliest infectious diseases, and Plasmodium, the causative agent, needs to complete a complex development cycle in its vector mosquito for transmission to occur. Two promising strategies to curb transmission are transgenesis, consisting of genetically engineering mosquitoes to express antimalarial effector molecules, and paratransgenesis, consisting of introducing into the mosquito commensal bacteria engineered to express antimalarial effector molecules. Although both approaches restrict parasite development in the mosquito, it is not known how their effectiveness compares. Here we provide an in-depth assessment of transgenesis and paratransgenesis and evaluate the combination of the two approaches. Using the Q-system to drive gene expression, we engineered mosquitoes to produce and secrete two effectors - scorpine and the MP2 peptide - into the mosquito gut and salivary glands. We also engineered Serratia, a commensal bacterium capable of spreading through mosquito populations to secrete effectors into the mosquito gut. Whereas both mosquito-based and bacteria-based approaches strongly reduced the oocyst and sporozoite intensity, a substantially stronger reduction of Plasmodium falciparum development was achieved when transgenesis and paratransgenesis were combined. Most importantly, transmission of Plasmodium berghei from infected to naïve mice was maximally inhibited by the combination of the two approaches. Combining these two strategies promises to become a powerful approach to combat malaria.

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.