Antibody-mediated immunity directed against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is an important immune mechanism for protection against clinical malaria and control of parasitemia. Current studies on PfEMP1-based immunity are constrained by the protein’s high molecular weight, complex multidomain architecture, and extensive polymorphism. Controlled human malaria infection (CHMI) studies uniquely enable the assessment of strain-specific PfEMP1 immunity prior to exposure and its correlation with infection outcomes using the same parasite strain. In this study, different serological assays targeting both full-length native PfEMP1 and individual domains were applied to plasma samples obtained prior to challenge infection at study baseline from 25 individuals enrolled in a CHMI study of PfSPZ Challenge (NF54), that included malaria-naïve Europeans and lifelong malaria-exposed Africans with varying degrees of immunity. All assays showed strong predictive value of both PfEMP1 antibody levels and breadth at study baseline for CHMI outcome. A random forest machine learning analysis of antibody recognition profiles, measured by AlphaScreen nearly covering all extracellular PfEMP1 domains of the NF54-CHMI parasite strain, suggested that a broad antibody repertoire, including antibodies against the most diversified group B PfEMP1s, represents a discriminative feature distinguishing volunteers with high versus low susceptibility to CHMI. In parasites re-isolated from the volunteers, dominant var transcripts encoded PfEMP1 variants are not recognized by the individuals’ pre-existing antibodies at study baseline. Given that the CHMI strain NF54 predominantly expresses B-type var genes at the onset of blood-stage infection, effective control of CHMI was associated with a broad antibody repertoire, particularly including antibodies targeting B-type PfEMP1. In line with the “hole-in-the-wall” theory, this likely reflects an antibody-mediated restriction of the parasite population emerging from the liver to parasites expressing PfEMP1 variants not previously encountered by the host immune system.

Malaria caused by Plasmodium falciparum remains a leading cause of childhood mortality in sub-Saharan Africa. The intraerythrocytic parasite can evade complement-mediated destruction through recruitment of human Factor H (FH). In vitro, Factor H-related protein 1 (FHR-1) competes with FH for binding sites, suggesting a potential role in modulating complement activation during infection. Because one-third of Africans carry a CFHR3/1 deletion, we examined the impact of FHR-1 deficiency on malaria severity. We analyzed plasma from 500 Ghanaian children presenting with different clinical manifestations. One-third of plasma samples lacked detectable FHR-1 protein, but logistic regression analysis revealed no association between FHR-1 deficiency and risk of severe malaria. In contrast, FHR-1 deficiency was overrepresented among children presenting with anemia and multiple syndromes and affected children exhibited markedly lower hemoglobin levels. These findings indicate that FHR-1 deficiency does not affect overall progression of severe falciparum malaria, but correlates with severe malarial anemia.

BACKGROUND:Schistosomiasis and malaria are major poverty-related parasitic infectious diseases. They are often coendemic and affect similar target populations. Mass drug administration and intermittent preventive treatment programs are commonly used for their control. From a programmatic perspective the establishment of preventive treatment programs targeting both diseases simultaneously would be a great advantage. While there is preliminary evidence that certain antimalarial drugs exert a clinically important effect on Schistosomes, little is known on whether praziquantel (PZQ), the only licensed drug to treat schistosomiasis, has activity against Plasmodium parasites. Therefore, the CORMA-MAL study was conducted to evaluate antimalarial activity of PZQ. METHODS:This double-blinded, randomized, placebo-controlled phase 2b trial was conducted in Lambaréné, Gabon and recruited semi-immune adults with an asymptomatic Plasmodium falciparum parasitemia. Participants were randomized (1:1) to receive placebo or 40 mg/kg PZQ once daily for 3 days and followed up for a total of 7 days. RESULTS:Forty-four participants were recruited. Analytical models indicated a highly significant hourly reduction of microscopically determined parasitemia in the PZQ arm and a nonsignificant decreasing trend in the placebo group (regression slopes of log10-transformed parasitemia: β=-0.006 [P < .001] and β=-0.0013 [P = .3], respectively). However, in total only 9 of 22 (41%) in the PZQ arm compared to 6 of 22 (27%) in the placebo arm reached parasite clearance by day 7. CONCLUSIONS:Findings demonstrate a statistically significant antimalarial activity of PZQ; however, the observed effect is only moderate compared to current first-line antimalarial treatments. Future research should investigate the synergistic potential of combining PZQ and antimalarial drugs in malaria and schistosomiasis control. Clinical Trials Registration.Pan African Clinical Trials Registry, PACTR202206584817951, 10 June 2022 (https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=22586).

The pathogenesis of severe Plasmodium falciparum malaria involves cytoadhesive microvascular sequestration of infected erythrocytes, mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 variants are encoded by the highly polymorphic family of var genes, the sequences of which are largely unknown in clinical samples. Previously, we published new approaches for var gene profiling and classification of predicted binding phenotypes in clinical P. falciparum isolates (Wichers et al., 2021), which represented a major technical advance. Building on this, we report here a novel method for var gene assembly and multidimensional quantification from RNA-sequencing that outperforms the earlier approach of Wichers et al., 2021, on both laboratory and clinical isolates across a combination of metrics. Importantly, the tool can interrogate the var transcriptome in context with the rest of the transcriptome and can be applied to enhance our understanding of the role of var genes in malaria pathogenesis. We applied this new method to investigate changes in var gene expression through early transition of parasite isolates to in vitro culture, using paired sets of ex vivo samples from our previous study, cultured for up to three generations. In parallel, changes in non-polymorphic core gene expression were investigated. Modest but unpredictable var gene switching and convergence towards var2csa were observed in culture, along with differential expression of 19% of the core transcriptome between paired ex vivo and generation 1 samples. Our results cast doubt on the validity of the common practice of using short-term cultured parasites to make inferences about in vivo phenotype and behaviour.

Volume 14, no. 1, e03318-23, 2023, https://doi.org/10.1128/mbio.03318-22. Page 10, Fig. 5A and B: Parts of the colored markups (yellow, green, and magenta) are missing. The panels should appear as shown below.

Controlled human malaria infections (CHMI) are a valuable tool to study parasite gene expression in vivo under defined conditions. In previous studies, virulence gene expression was analyzed in samples from volunteers infected with the Plasmodium falciparum (Pf) NF54 isolate, which is of African origin. Here, we provide an in-depth investigation of parasite virulence gene expression in malaria-naïve European volunteers undergoing CHMI with the genetically distinct Pf 7G8 clone, originating in Brazil. Differential expression of var genes, encoding major virulence factors of Pf, PfEMP1s, was assessed in ex vivo parasite samples as well as in parasites from the in vitro cell bank culture that was used to generate the sporozoites (SPZ) for CHMI (Sanaria PfSPZ Challenge (7G8)). We report broad activation of mainly B-type subtelomeric located var genes at the onset of a 7G8 blood stage infection in naïve volunteers, mirroring the NF54 expression study and suggesting that the expression of virulence-associated genes is generally reset during transmission from the mosquito to the human host. However, in 7G8 parasites, we additionally detected a continuously expressed single C-type variant, Pf7G8_040025600, that was most highly expressed in both pre-mosquito cell bank and volunteer samples, suggesting that 7G8, unlike NF54, maintains expression of some previously expressed var variants during transmission. This suggests that in a new host, the parasite may preferentially express the variants that previously allowed successful infection and transmission. Trial registration: ClinicalTrials.gov - NCT02704533 ; 2018-004523-36

Mature gametocytes of Plasmodium falciparum display a banana (falciform) shape conferred by a complex array of subpellicular microtubules (SPMT) associated with the inner membrane complex (IMC). Microtubule-associated proteins (MAPs) define MT populations and modulate interaction with pellicular components. Several MAPs have been identified in Toxoplasma gondii, and homologues can be found in the genomes of Plasmodium species, but the function of these proteins for asexual and sexual development of malaria parasites is still unknown. Here, we identified a novel subpellicular MAP, termed SPM3, that is conserved within the genus Plasmodium, especially within the subgenus Laverania, but absent in other Apicomplexa. Conditional knockdown and targeted gene disruption of Pfspm3 in Plasmodium falciparum cause severe morphological defects during gametocytogenesis, leading to round, nonfalciform gametocytes with an aberrant SPMT pattern. In contrast, Pbspm3 knockout in Plasmodium berghei, a species with round gametocytes, caused no defect in gametocytogenesis, but sporozoites displayed an aberrant motility and a dramatic defect in invasion of salivary glands, leading to a decreased efficiency in transmission. Electron microscopy revealed a dissociation of the SPMT from the IMC in Pbspm3 knockout parasites, suggesting a function of SPM3 in anchoring MTs to the IMC. Overall, our results highlight SPM3 as a pellicular component with essential functions for malaria parasite transmission. IMPORTANCE A key structural feature driving the transition between different life cycle stages of the malaria parasite is the unique three-membrane pellicle, consisting of the parasite plasma membrane (PPM) and a double membrane structure underlying the PPM termed the inner membrane complex (IMC). Additionally, there are numerous linearly arranged intramembranous particles (IMPs) linked to the IMC, which likely link the IMC to the subpellicular microtubule cytoskeleton. Here, we identified, localized, and characterized a novel subpellicular microtubule-associated protein unique to the genus Plasmodium. The knockout of this protein in the human-pathogenic species P. falciparum resulted in malformed gametocytes and aberrant microtubules. We confirmed the microtubule association in the P. berghei rodent malaria homologue and show that its knockout results in a perturbed microtubule architecture, aberrant sporozoite motility, and decreased transmission efficiency.

AbstractThe pathogenesis of severePlasmodium falciparummalaria involves cytoadhesive microvascular sequestration of infected erythrocytes, mediated byP. falciparumerythrocyte membrane protein 1 (PfEMP1). PfEMP1 variants are encoded by the highly polymorphic family ofvargenes, the sequences of which are largely unknown in clinical samples. Previously, we published new approaches forvargene profiling and classification of predicted binding phenotypes in clinicalP. falciparumisolates (Wicherset al., 2021), which represented a major technical advance. Building on this, we report here a novel method forvargene assembly and multidimensional quantification from RNA-sequencing that outperforms the earlier approach of Wicherset al., 2021 on both laboratory and clinical isolates across a combination of metrics. Importantly, the tool can interrogate thevartranscriptome in context with the rest of the transcriptome and can be applied to enhance our understanding of the role ofvargenes in malaria pathogenesis. We applied this new method to investigate changes invargene expression through early transition of parasite isolates toin vitroculture, using paired sets ofex vivosamples from our previous study, cultured for up to three generations. In parallel, changes in non-polymorphic core gene expression were investigated. Modest but unpredictablevargene switching and convergence towardsvar2csawere observed in culture, along with differential expression of 19% of the core transcriptome between pairedex vivoand generation 1 samples. Our results cast doubt on the validity of the common practice of using short-term cultured parasites to make inferences aboutin vivophenotype and behaviour.

Plasmodium falciparum-infected erythrocytes (PfIEs) present P. falciparum erythrocyte membrane protein 1 proteins (PfEMP1s) on the cell surface, via which they cytoadhere to various endothelial cell receptors (ECRs) on the walls of human blood vessels. This prevents the parasite from passing through the spleen, which would lead to its elimination. Each P. falciparum isolate has about 60 different PfEMP1s acting as ligands, and at least 24 ECRs have been identified as interaction partners. Interestingly, in every parasite genome sequenced to date, at least 75% of the encoded PfEMP1s have a binding domain for the scavenger receptor CD36 widely distributed on host endothelial cells and many other cell types. Here, we discuss why the interaction between PfIEs and CD36 is optimal to maintain a finely regulated equilibrium that allows the parasite to multiply and spread while causing minimal harm to the host in most infections.

Quantitative real-time PCR (qPCR) is a simple and sensitive method for determining the amount of a specific target DNA sequence present in a sample. Compared to RNA-seq, reverse transcription qPCR (RT-qPCR) is fast, requires only low input material and is easy to analyze. Therefore, qPCR is widely used to analyze gene expression in P. falciparum, including analyses of the multicopy gene families encoding variant surface antigens (VSAs), whose expression is clonally variant and prone to changes over time. In the recent years, several P. falciparum genomes of culture-adapted strains have been sequenced, providing the knowledge to design variable gene family-specific qPCR primers for each P. falciparum genetic background. Here, we describe the required materials, methods and key factors to perform RT-qPCR experiments to determine VSA transcript abundances in the P. falciparum clones 3D7/NF54, IT4, HB3, and 7G8.

Malaria is responsible for hundreds of thousands of deaths every year. The lack of an effective vaccine and the global spread of multidrug resistant parasites hampers the fight against the disease and underlines the need for new antimalarial drugs. Central to the pathogenesis of malaria is the proliferation of Plasmodium parasites within human erythrocytes. Parasites invade erythrocytes via a coordinated sequence of receptor-ligand interactions between the parasite and the host cell. Posttranslational modifications such as protein phosphorylation are known to be key regulators in this process and are mediated by protein kinases. For several parasite kinases, including the Plasmodium falciparum glycogen synthase kinase 3 (PfGSK3), inhibitors have been shown to block erythrocyte invasion. Here, we provide an assessment of PfGSK3 function by reverse genetics. Using targeted gene disruption, we show the active gene copy, PfGSK3β, is not essential for asexual blood stage proliferation, although it modulates efficient erythrocyte invasion. We found functional inactivation leads to a 69% decreased growth rate and confirmed this growth defect by rescue experiments with wildtype and catalytically inactive mutants. Functional knockout of PfGSK3β does not lead to transcriptional upregulation of the second copy of PfGSK3. We further analyze expression, localization, and function of PfGSK3β during gametocytogenesis using a parasite line allowing conditional induction of sexual commitment. We demonstrate PfGSK3β-deficient gametocytes show a strikingly malformed morphology leading to the death of parasites in later stages of gametocyte development. Taken together, these findings are important for our understanding and the development of PfGSK3 as an antimalarial target.

Epigenetic regulation is a critical mechanism in controlling virulence, differentiation, and survival of the human malaria parasite Plasmodium (P.) falciparum. Bromodomain proteins contribute to this process by binding to acetylated lysine residues of histones and thereby targeting the gene regulatory machinery to gene promoters. A protein complex containing the P. falciparum bromodomain proteins (PfBDP) 1 and PfBDP2 (BDP1/BDP2 core complex) was previously shown to play an essential role for the correct transcription of invasion related genes. Here, we performed a functional characterization of a third component of this complex, which we dubbed PfBDP7, because structural modelling predicted a typical bromodomain fold. We confirmed that PfBDP7 is a nuclear protein that interacts with PfBDP1 at invasion gene promoters in mature schizont stage parasites and contributes to their transcription. Although partial depletion of PfBDP7 showed no significant effect on parasite viability, conditional knock down of either PfBDP7 or PfBDP1 resulted in the de-repression of variant surface antigens (VSA), which are important pathogenicity factors. This de-repression was evident both on mRNA and protein level. To understand the underlying mechanism, we mapped the genome wide binding sites of PfBDP7 by ChIPseq and showed that in early schizonts, PfBDP7 and PfBDP1 are commonly enriched in heterochromatic regions across the gene body of all VSA families, including genes coding for PfEMP1, RIFIN, STEVOR, and PfMC-2TM. This suggests that PfBDP7 and PfBDP1 contribute to the silencing of VSAs by associating with heterochromatin. In conclusion, we identified PfBDP7 as a chromatin binding protein that is a constitutive part of the P. falciparum BDP1/BDP2 core complex and established PfBDP1 and PfBDP7 as novel players in the silencing of heterochromatin regulated virulence gene families of the malaria parasite P. falciparum.

Membrane transport proteins perform crucial roles in cell physiology. The obligate intracellular parasite Plasmodium falciparum, an agent of human malaria, relies on membrane transport proteins for the uptake of nutrients from the host, disposal of metabolic waste, exchange of metabolites between organelles, and generation and maintenance of transmembrane electrochemical gradients for its growth and replication within human erythrocytes. Despite their importance for Plasmodium cellular physiology, the functional roles of a number of membrane transport proteins remain unclear, which is particularly true for orphan membrane transporters that have no or limited sequence homology to transporter proteins in other evolutionary lineages. Therefore, in the current study, we applied endogenous tagging, targeted gene disruption, conditional knockdown, and knockout approaches to investigate the subcellular localization and essentiality of six membrane transporters during intraerythrocytic development of P. falciparum parasites. They are localized at different subcellular structures-the food vacuole, the apicoplast, and the parasite plasma membrane-and four out of the six membrane transporters are essential during asexual development. Additionally, the plasma membrane resident transporter 1 (PMRT1; PF3D7_1135300), a unique Plasmodium-specific plasma membrane transporter, was shown to be essential for gametocytogenesis and functionally conserved within the genus Plasmodium. Overall, we reveal the importance of four orphan transporters to blood stage P. falciparum development, which have diverse intracellular localizations and putative functions. IMPORTANCE Plasmodium falciparum-infected erythrocytes possess multiple compartments with designated membranes. Transporter proteins embedded in these membranes not only facilitate movement of nutrients, metabolites, and other molecules between these compartments, but also are common therapeutic targets and can confer antimalarial drug resistance. Orphan membrane transporters in P. falciparum without sequence homology to transporters in other evolutionary lineages and divergent from host transporters may constitute attractive targets for novel intervention approaches. Here, we localized six of these putative transporters at different subcellular compartments and probed their importance during asexual parasite growth by using reverse genetic approaches. In total, only two candidates turned out to be dispensable for the parasite, highlighting four candidates as putative targets for therapeutic interventions. This study reveals the importance of several orphan transporters to blood stage P. falciparum development.

Sequestration of Plasmodium falciparum -infected erythrocytes to host endothelium through the parasite-derived Pf EMP1 adhesion proteins is central to the development of malaria pathogenesis. Pf EMP1 proteins have diversified and expanded to encompass many sequence variants conferring each parasite a similar array of human endothelial receptor binding phenotypes. Here, we analyzed RNA-seq profiles of parasites isolated from 32 P. falciparum infected adult travelers returning to Germany. Patients were categorized into either malaria naïve (n=15) or pre-exposed (n=17), and into severe (n=8) or non-severe (n=24) cases. For differential expression analysis of Pf EMP1-encoding var gene transcripts were de novo assembled from RNA-seq data and, in parallel, var expressed sequence tags were analyzed and used to predict the encoded domain composition of the transcripts. Both approaches showed in concordance that severe malaria was associated with Pf EMP1 containing the endothelial protein C receptor (EPCR)-binding CIDRα1 domain, whereas CD36-binding Pf EMP1 was linked to non-severe malaria outcomes. First-time infected adults were more likely to develop severe symptoms and tended to be infected for a longer period. Thus, parasites with more pathogenic Pf EMP1 variants are more common in patients with a naïve immune status and/or adverse inflammatory host responses to first infections favors growth of EPCR-binding parasites. ### Competing Interest Statement The authors have declared no competing interest.

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.

Sequestration of Plasmodium falciparum(P. falciparum)-infected erythrocytes to host endothelium through the parasite-derived P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion proteins is central to the development of malaria pathogenesis. PfEMP1 proteins have diversified and expanded to encompass many sequence variants, conferring each parasite a similar array of human endothelial receptor-binding phenotypes. Here, we analyzed RNA-seq profiles of parasites isolated from 32 P. falciparum-infected adult travellers returning to Germany. Patients were categorized into either malaria naive (n = 15) or pre-exposed (n = 17), and into severe (n = 8) or non-severe (n = 24) cases. For differential expression analysis, PfEMP1-encoding var gene transcripts were de novo assembled from RNA-seq data and, in parallel, var-expressed sequence tags were analyzed and used to predict the encoded domain composition of the transcripts. Both approaches showed in concordance that severe malaria was associated with PfEMP1 containing the endothelial protein C receptor (EPCR)-binding CIDRα1 domain, whereas CD36-binding PfEMP1 was linked to non-severe malaria outcomes. First-time infected adults were more likely to develop severe symptoms and tended to be infected for a longer period. Thus, parasites with more pathogenic PfEMP1 variants are more common in patients with a naive immune status, and/or adverse inflammatory host responses to first infections favor the growth of EPCR-binding parasites.

Intraerythrocytic malaria parasites proliferate bounded by a parasitophorous vacuolar membrane (PVM). The PVM contains nutrient permeable channels (NPCs) conductive to small molecules, but their relevance for parasite growth for individual metabolites is largely untested. Here we show that growth-relevant levels of major carbon and energy sources pass through the NPCs. Moreover, we find that NPCs are a gate for several antimalarial drugs, highlighting their permeability properties as a critical factor for drug design. Looking into NPC-dependent amino acid transport, we find that amino acid shortage is a reason for the fitness cost in artemisinin-resistant (ARTR) parasites and provide evidence that NPC upregulation to increase amino acids acquisition is a mechanism of ARTR parasites in vitro and in human infections to compensate this fitness cost. Hence, the NPCs are important for nutrient and drug access and reveal amino acid deprivation as a critical constraint in ARTR parasites.