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The malaria parasites Plasmodium falciparum and Plasmodium vivax differ in key biological processes and associated clinical effects, but consequences on population-level transmission dynamics are difficult to predict. This co-endemic malaria study from Guyana details important epidemiological contrasts between the species by coupling population genomics (1396 spatiotemporally matched parasite genomes, primarily from 2020–21) with sociodemographic analysis (nationwide patient census from 2019). We describe how P. falciparum forms large, interrelated subpopulations that sporadically expand but generally exhibit restrained dispersal, whereby spatial distance and patient travel statistics predict parasite identity-by-descent (IBD). Case bias towards working-age adults is also strongly pronounced. P. vivax exhibits 46% higher average nucleotide diversity (π) and 6.5x lower average IBD. It occupies a wider geographic range, without evidence for outbreak-like expansions, only microgeographic patterns of isolation-by-distance, and weaker case bias towards adults. Possible latency-relapse effects also manifest in various analyses. For example, 11.0% of patients diagnosed with P. vivax in Greater Georgetown report no recent travel to endemic zones, and P. vivax clones recur in 11 of 46 patients incidentally sampled twice during the study. Polyclonality rate is also 2.1x higher than in P. falciparum, does not trend positively with estimated incidence, and correlates uniquely to selected demographics. We discuss possible underlying mechanisms and implications for malaria control. P. falciparum and vivax are responsible for most cases of malaria but are not genetically closely related and differ in their clinical and epidemiological impacts. In this study, the authors investigate the genomic and epidemiological characteristics of the two parasites in a co-endemic setting of Guyana. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum is the main causative agent of malaria, a deadly disease that mainly affects children under five years old. Artemisinin-based combination therapies have been pivotal in controlling the disease, but resistance has arisen in various regions, increasing the risk of treatment failure. The non-mevalonate pathway is essential for the isoprenoid synthesis in Plasmodium and provides several under-explored targets to be used in the discovery of new antimalarials. 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) is the first and rate-limiting enzyme of the pathway. Despite its importance, there are no structures available for any Plasmodium spp., due to the complex sequence which contains large regions of high disorder, making crystallisation a difficult task. In this manuscript, we use cryo-electron microscopy to solve the P. falciparum DXPS structure at a final resolution of 2.42 Å. Overall, the structure resembles other DXPS enzymes but includes a distinct N-terminal domain exclusive to the Plasmodium genus. Mutational studies show that destabilization of the cap domain interface negatively impacts protein stability and activity. Additionally, a density for the co-factor thiamine diphosphate is found in the active site. Our work highlights the potential of cryo-EM to obtain structures of P. falciparum proteins that are unfeasible by means of crystallography. DXPS is an important enzyme for isoprenoid synthesis in Plasmodium falciparum. Here, authors elucidate the cryo-EM structure of PfDXPS showing an N-terminal domain only present in this genus. Mutation studies show its importance in DXPS stability and activity. [ABSTRACT FROM AUTHOR]

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. Maurizio and Masid et al. identify common host metabolic pathways essential for Plasmodium falciparum and Theileria parasites, revealing potential for developing shared broad-spectrum antiparasitic therapies to more effectively combat these debilitating infections. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum is the causative agent of malaria and remains a pathogen of global importance. Asexual blood stage replication, via a process called schizogony, is an important target for the development of new antimalarials. Here we use ultrastructure-expansion microscopy to probe the organisation of the chromosome-capturing kinetochores in relation to the mitotic spindle, the centriolar plaque, the centromeres and the apical organelles during schizont development. Conditional disruption of the kinetochore components, PfNDC80 and PfNuf2, is associated with aberrant mitotic spindle organisation, disruption of the centromere marker, CENH3 and impaired karyokinesis. Surprisingly, kinetochore disruption also leads to disengagement of the centrosome equivalent from the nuclear envelope. Severing the connection between the nucleus and the apical complex leads to the formation of merozoites lacking nuclei. Here, we show that correct assembly of the kinetochore/spindle complex plays a previously unrecognised role in positioning the nascent apical complex in developing P. falciparum merozoites. Using Ultra-Expansion Microscopy of the malaria parasite, P. falciparum, Li et al. show that disruption of kinetochore components breaks a nexus between the mitotic spindle and the nascent apical organelles. [ABSTRACT FROM AUTHOR]

Host immune responses are tightly controlled by various immune factors during infection, and protozoan parasites also manipulate the immune system to evade surveillance, leading to an evolutionary arms race in host‒pathogen interactions; however, the underlying mechanisms are not fully understood. We observed that the level of superoxide dismutase 3 (SOD3) was significantly elevated in both Plasmodium falciparum malaria patients and mice infected with four parasite species. SOD3-deficient mice had a substantially longer survival time and lower parasitemia than control mice after infection, whereas SOD3-overexpressing mice were much more vulnerable to parasite infection. We revealed that SOD3, secreted from activated neutrophils, bound to T cells, suppressed the interleukin-2 expression and concomitant interferon-gamma responses crucial for parasite clearance. Overall, our findings expose active fronts in the arms race between the parasites and host immune system and provide insights into the roles of SOD3 in shaping host innate immune responses to parasite infection. Superoxide dismutase 3 is elevated in patients and animals infected by protozoan parasites such as Plasmodium. Here, Li et al. show that SOD3 expression is associated with experimental cerebral malaria and inhibition of host immune responses. [ABSTRACT FROM AUTHOR]

Reticulocyte-binding protein homologue 5 (RH5), a leading blood-stage Plasmodium falciparum malaria vaccine target, interacts with cysteine-rich protective antigen (CyRPA) and RH5-interacting protein (RIPR) to form an essential heterotrimeric "RCR-complex". We investigate whether RCR-complex vaccination can improve upon RH5 alone. Using monoclonal antibodies (mAbs) we show that parasite growth-inhibitory epitopes on each antigen are surface-exposed on the RCR-complex and that mAb pairs targeting different antigens can function additively or synergistically. However, immunisation of female rats with the RCR-complex fails to outperform RH5 alone due to immuno-dominance of RIPR coupled with inferior potency of anti-RIPR polyclonal IgG. We identify that all growth-inhibitory antibody epitopes of RIPR cluster within the C-terminal EGF-like domains and that a fusion of these domains to CyRPA, called "R78C", combined with RH5, improves the level of in vitro parasite growth inhibition compared to RH5 alone. These preclinical data justify the advancement of the RH5.1 + R78C/Matrix-M™ vaccine candidate to Phase 1 clinical trial. RH5, which is part of the trimeric RCR-complex essential for invasion, is a vaccine candidate for malaria. Here, Williams et al. show that monoclonal antibodies targeting each of the three proteins in the RCR-complex can work together to more effectively block the invasion of red blood cells by Plasmodium falciparum and design a combination vaccine candidate. [ABSTRACT FROM AUTHOR]

Despite concern that climate change could increase the human risk to malaria in certain areas, the temperature dependency of malaria transmission is poorly characterized. Here, we use a mechanistic model fitted to experimental data to describe how Plasmodium falciparum infection of the African malaria vector, Anopheles gambiae, is modulated by temperature, including its influences on parasite establishment, conversion efficiency through parasite developmental stages, parasite development rate, and overall vector competence. We use these data, together with estimates of the survival of infected blood-fed mosquitoes, to explore the theoretical influence of temperature on transmission in four locations in Kenya, considering recent conditions and future climate change. Results provide insights into factors limiting transmission in cooler environments and indicate that increases in malaria transmission due to climate warming in areas like the Kenyan Highlands, might be less than previously predicted. Malaria transmission is affected by temperature but this relationship is not well characterised. Here, the authors experimentally determine the effect of temperature on parasite development in the mosquito and model how it impacts malaria transmission in Kenya under current and future climate scenarios. [ABSTRACT FROM AUTHOR]

Routine sampling of pregnant women at first antenatal care (ANC) visits could make Plasmodium falciparum genomic surveillance more cost-efficient and convenient in sub-Saharan Africa. We compare the genetic structure of parasite populations sampled from 289 first ANC users and 93 children from the community in Mozambique between 2015 and 2019. Samples are amplicon sequenced targeting 165 microhaplotypes and 15 drug resistance genes. Metrics of genetic diversity and relatedness, as well as the prevalence of drug resistance markers, are consistent between the two populations. In an area targeted for elimination, intra-host genetic diversity declines in both populations (p = 0.002-0.007), while for the ANC population, population genetic diversity is also lower (p = 0.0004), and genetic relatedness between infections is higher (p = 0.002) than control areas, indicating a recent reduction in the parasite population size. These results highlight the added value of genomic surveillance at ANC clinics to inform about changes in transmission beyond epidemiological data. Routine sampling of pregnant women at first antenatal care (ANC) visits could be used for malaria surveillance. Here, the authors compare the genetic structure of Plasmodium falciparum parasite populations between samples from first ANC users and children from the community in Mozambique, and show that it can inform about changes in transmission beyond epidemiological data. [ABSTRACT FROM AUTHOR]

Delaying and slowing antimalarial drug resistance evolution is a priority for malaria-endemic countries. Until novel therapies become available, the mainstay of antimalarial treatment will continue to be artemisinin-based combination therapy (ACT). Deployment of different ACTs can be optimized to minimize evolutionary pressure for drug resistance by deploying them as a set of co-equal multiple first-line therapies (MFT) rather than rotating therapies in and out of use. Here, we consider one potential detriment of MFT policies, namely, that the simultaneous deployment of multiple ACTs could drive the evolution of different resistance alleles concurrently and that these resistance alleles could then be brought together by recombination into double-resistant or triple-resistant parasites. Using an individual-based model, we compare MFT and cycling policies in malaria transmission settings ranging from 0.1% to 50% prevalence. We define a total risk measure for multi-drug resistance (MDR) by summing the area under the genotype-frequency curves (AUC) of double- and triple-resistant genotypes. When prevalence ≥ 1%, total MDR risk ranges from statistically similar to 80% lower under MFT policies than under cycling policies, irrespective of whether resistance is imported or emerges de novo. At 0.1% prevalence, there is little statistical difference in MDR risk between MFT and cycling. Emergence of malaria parasites resistant to artemisinin has prompted the need for new drug regimens to ensure effective treatment. In this simulation study, the authors evaluate the risk of multidrug resistance under regimens with either concurrent or cyclic use of different first-line therapies. [ABSTRACT FROM AUTHOR]

The proteasome of the malaria parasite Plasmodium falciparum (Pf20S) is an advantageous drug target because its inhibition kills P. falciparum in multiple stages of its life cycle and synergizes with artemisinins. We recently developed a macrocyclic peptide, TDI-8304, that is highly selective for Pf20S over human proteasomes and is potent in vitro and in vivo against P. falciparum. A mutation in the Pf20S β6 subunit, A117D, confers resistance to TDI-8304, yet enhances both enzyme inhibition and anti-parasite activity of a tripeptide vinyl sulfone β2 inhibitor, WLW-vs. Here we present the high-resolution cryo-EM structures of Pf20S with TDI-8304, of human constitutive proteasome with TDI-8304, and of Pf20Sβ6A117D with WLW-vs that give insights into the species selectivity of TDI-8304, resistance to it, and the collateral sensitivity associated with resistance, including that TDI-8304 binds β2 and β5 in wild type Pf20S as well as WLW-vs binds β2 and β5 in Pf20Sβ6A117D. We further show that TDI-8304 kills P. falciparum as quickly as chloroquine and artemisinin and is active against P. cynomolgi at the liver stage. This increases interest in using these structures to facilitate the development of Pf20S inhibitors that target multiple proteasome subunits and limit the emergence of resistance. The proteasome of Plasmodium falciparum (Pf20S) is a promising multi-stage drug target. Using CryoEM Hsu et al. report the structures of parasite and human proteasome in complex with the recently developed inhibitor TDI-8304 to gain insights into its species selectivity. [ABSTRACT FROM AUTHOR]

Clinical immunity against Plasmodium falciparum infection develops in residents of malaria endemic regions, manifesting in reduced clinical symptoms during infection and in protection against severe disease but the mechanisms are not fully understood. Here, we compare the cellular and humoral immune response of clinically immune (0-1 episode over 18 months) and susceptible (at least 3 episodes) during a mild episode of Pf malaria infection in a malaria endemic region of Malawi, by analysing peripheral blood samples using high dimensional mass cytometry (CyTOF), spectral flow cytometry and single-cell transcriptomic analyses. In the clinically immune, we find increased proportions of circulating follicular helper T cells and classical monocytes, while the humoral immune response shows characteristic age-related differences in the protected. Presence of memory CD4+ T cell clones with a strong cytolytic ZEB2+ T helper 1 effector signature, sharing identical T cell receptor clonotypes and recognizing the Pf-derived circumsporozoite protein (CSP) antigen are found in the blood of the Pf-infected participants gaining protection. Moreover, in clinically protected participants, ZEB2+ memory CD4+ T cells express lower level of inhibitory and chemotactic receptors. We thus propose that clonally expanded ZEB2+ CSP-specific cytolytic memory CD4+ Th1 cells may contribute to clinical immunity against the sporozoite and liver-stage Pf malaria. It is important to understand why some individuals in endemic regions acquire natural immunity against malaria while others remain susceptible. Here authors show that during episodes of Plasmodium falciparum malaria, circumsporozoite-specific cytolytic memory CD4+ T cells are clonally expanded in patients, and those with clinical immunity demonstrate reduction in the chemotactic and inhibitory receptor expression in ZEB2+ memory CD4+ T cells. [ABSTRACT FROM AUTHOR]

We here analyze data from the first year of an ongoing nationwide program of genetic surveillance of Plasmodium falciparum parasites in Senegal. The analysis is based on 1097 samples collected at health facilities during passive malaria case detection in 2019; it provides a baseline for analyzing parasite genetic metrics as they vary over time and geographic space. The study's goal was to identify genetic metrics that were informative about transmission intensity and other aspects of transmission dynamics, focusing on measures of genetic relatedness between parasites. We found the best genetic proxy for local malaria incidence to be the proportion of polygenomic infections (those with multiple genetically distinct parasites), although this relationship broke down at low incidence. The proportion of related parasites was less correlated with incidence while local genetic diversity was uninformative. The type of relatedness could discriminate local transmission patterns: two nearby areas had similarly high fractions of relatives, but one was dominated by clones and the other by outcrossed relatives. Throughout Senegal, 58% of related parasites belonged to a single network of relatives, within which parasites were enriched for shared haplotypes at known and suspected drug resistance loci and at one novel locus, reflective of ongoing selection pressure. Senegal has initiated a national sentinel surveillance program for malaria parasite genetics. Here, the authors report data from the first year of the program and use it to investigate local malaria incidence, patterns of transmission, and genetic loci under selection. [ABSTRACT FROM AUTHOR]

Placental malaria is caused by Plasmodium falciparum-infected erythrocytes (IEs) adhering to chondroitin sulfate proteoglycans in placenta via VAR2CSA-type PfEMP1. Human pentameric immunoglobulin M (IgM) binds to several types of PfEMP1, including VAR2CSA via its Fc domain. Here, a 3.6 Å cryo-electron microscopy map of the IgM-VAR2CSA complex reveals that two molecules of VAR2CSA bind to the Cµ4 of IgM through their DBL3X and DBL5ε domains. The clockwise and anti-clockwise rotation of the two VAR2CSA molecules on opposite faces of IgM juxtaposes C-termini of both VAR2CSA near the J chain, where IgM creates a wall between both VAR2CSA molecules and hinders its interaction with its receptor. To support this, we show when VAR2CSA is bound to IgM, its staining on IEs as well as binding of IEs to chondroitin sulfate A in vitro is severely compromised. Placental malaria parasite binds to host IgM however its functional importance was not understood. Here, authors resolved structure of IgM-VAR2CSA complex using cryo-EM to show that IgM masks VAR2CSA and inhibits its binding to receptor. [ABSTRACT FROM AUTHOR]

We report an analysis of the propensity of the antimalarial agent cabamiquine, a Plasmodium-specific eukaryotic elongation factor 2 inhibitor, to select for resistant Plasmodium falciparum parasites. Through in vitro studies of laboratory strains and clinical isolates, a humanized mouse model, and volunteer infection studies, we identified resistance-associated mutations at 11 amino acid positions. Of these, six (55%) were present in more than one infection model, indicating translatability across models. Mathematical modelling suggested that resistant mutants were likely pre-existent at the time of drug exposure across studies. Here, we estimated a wide range of frequencies of resistant mutants across the different infection models, much of which can be attributed to stochastic differences resulting from experimental design choices. Structural modelling implicates binding of cabamiquine to a shallow mRNA binding site adjacent to two of the most frequently identified resistance mutations. Authors utilize a number of models (mathematical, in vitro and in vivo infection) to analyse pre-clinical and Phase I clinical trial data, in regard to potential risk of resistance associated with a Plasmodium falciparum inhibitor, cabamiquine. [ABSTRACT FROM AUTHOR]

Apical membrane antigen 1 (AMA1) is a key malaria vaccine candidate and target of neutralizing antibodies. AMA1 binds to a loop in rhoptry neck protein 2 (RON2L) to form the moving junction during parasite invasion of host cells, and this complex is conserved among apicomplexan parasites. AMA1-RON2L complex immunization achieves higher growth inhibitory activity than AMA1 alone and protects mice against Plasmodium yoelii challenge. Here, three single-component AMA1-RON2L immunogens were designed that retain the structure of the two-component AMA1-RON2L complex: one structure-based design (SBD1) and two insertion fusions. All immunogens elicited high antibody titers with potent growth inhibitory activity, yet these antibodies did not block RON2L binding to AMA1. The SBD1 immunogen induced significantly more potent strain-transcending neutralizing antibody responses against diverse strains of Plasmodium falciparum than AMA1 or AMA1-RON2L complex vaccination. This indicates that SBD1 directs neutralizing antibody responses to strain-transcending epitopes in AMA1 that are independent of RON2L binding. This work underscores the importance of neutralization mechanisms that are distinct from RON2 blockade. The stable single-component SBD1 immunogen elicits potent strain-transcending protection that may drive the development of next-generation vaccines for improved malaria and apicomplexan parasite control. Here the authors use structure-based design to engineer a single component immunogen that mimics the malaria parasite AMA1-RON2 complex required for invasion of host cells, and show that it elicits a potent strain-transcending antibody response in rats. [ABSTRACT FROM AUTHOR]

We report an analysis of the propensity of the antimalarial agent cabamiquine, a Plasmodium-specific eukaryotic elongation factor 2 inhibitor, to select for resistant Plasmodium falciparum parasites. Through in vitro studies of laboratory strains and clinical isolates, a humanized mouse model, and volunteer infection studies, we identified resistance-associated mutations at 11 amino acid positions. Of these, six (55%) were present in more than one infection model, indicating translatability across models. Mathematical modelling suggested that resistant mutants were likely pre-existent at the time of drug exposure across studies. Here, we estimated a wide range of frequencies of resistant mutants across the different infection models, much of which can be attributed to stochastic differences resulting from experimental design choices. Structural modelling implicates binding of cabamiquine to a shallow mRNA binding site adjacent to two of the most frequently identified resistance mutations. Authors utilize a number of models (mathematical, in vitro and in vivo infection) to analyse pre-clinical and Phase I clinical trial data, in regard to potential risk of resistance associated with a Plasmodium falciparum inhibitor, cabamiquine. [ABSTRACT FROM AUTHOR]

The complex life cycle of Plasmodium falciparum requires coordinated gene expression regulation to allow host cell invasion, transmission, and immune evasion. Increasing evidence now suggests a major role for epigenetic mechanisms in gene expression in the parasite. In eukaryotes, many lncRNAs have been identified to be pivotal regulators of genome structure and gene expression. To investigate the regulatory roles of lncRNAs in P. falciparum we explore the intergenic lncRNA distribution in nuclear and cytoplasmic subcellular locations. Using nascent RNA expression profiles, we identify a total of 1768 lncRNAs, of which 718 (~41%) are novels in P. falciparum. The subcellular localization and stage-specific expression of several putative lncRNAs are validated using RNA-FISH. Additionally, the genome-wide occupancy of several candidate nuclear lncRNAs is explored using ChIRP. The results reveal that lncRNA occupancy sites are focal and sequence-specific with a particular enrichment for several parasite-specific gene families, including those involved in pathogenesis and sexual differentiation. Genomic and phenotypic analysis of one specific lncRNA demonstrate its importance in sexual differentiation and reproduction. Our findings bring a new level of insight into the role of lncRNAs in pathogenicity, gene regulation and sexual differentiation, opening new avenues for targeted therapeutic strategies against the deadly malaria parasite. In eukaryotes, long non-coding RNAs (lncRNAs) are regulators of gene expression. Here the authors identify lncRNAs implicated in pathogenicity and sexual differentiation of the human malaria parasite, P. falciparum, opening novel avenues towards therapeutic strategies. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum (Pf) parasite development in liver represents the initial step of the life-cycle in the human host after a Pf-infected mosquito bite. While an attractive stage for life-cycle interruption, understanding of parasite-hepatocyte interaction is inadequate due to limitations of existing in vitro models. We explore the suitability of hepatocyte organoids (HepOrgs) for Pf-development and show that these cells permitted parasite invasion, differentiation and maturation of different Pf strains. Single-cell messenger RNA sequencing (scRNAseq) of Pf-infected HepOrg cells has identified 80 Pf-transcripts upregulated on day 5 post-infection. Transcriptional profile changes are found involving distinct metabolic pathways in hepatocytes with Scavenger Receptor B1 (SR-B1) transcripts highly upregulated. A novel functional involvement in schizont maturation is confirmed in fresh primary hepatocytes. Thus, HepOrgs provide a strong foundation for a versatile in vitro model for Pf liver-stages accommodating basic biological studies and accelerated clinical development of novel tools for malaria control. Suitable in vitro models allowing to assess Plasmodium liver stage development are still limited. Here, Yang et al. show that hepatocytes derived from human hepatocyte organoids (HepOrgs) can support P. falciparum development. This allowed for the identification and validation of the importance of the host factor, scavenger receptor B1 (SRB1), in parasite development. [ABSTRACT FROM AUTHOR]

The chloroquine resistance transporter, PfCRT, of the human malaria parasite Plasmodium falciparum is sensitive to acidic pH. Consequently, PfCRT operates at 60% of its maximal drug transport activity at the pH of 5.2 of the digestive vacuole, a proteolytic organelle from which PfCRT expels drugs interfering with heme detoxification. Here we show by alanine-scanning mutagenesis that E207 is critical for pH sensing. The E207A mutation abrogates pH-sensitivity, while preserving drug substrate specificity. Substituting E207 with Asp or His, but not other amino acids, restores pH-sensitivity. Molecular dynamics simulations and kinetics analyses suggest an allosteric binding model in which PfCRT can accept both protons and chloroquine in a partial noncompetitive manner, with increased proton concentrations decreasing drug transport. Further simulations reveal that E207 relocates from a peripheral to an engaged location during the transport cycle, forming a salt bridge with residue K80. We propose that the ionized carboxyl group of E207 acts as a hydrogen acceptor, facilitating transport cycle progression, with pH sensing as a by-product. PfCRT is a chloroquine resistance transporter from malaria parasite Plasmodium falciparum, which is sensitive to acidic pH. Here, the authors show that residue E207 is critical for pH sensing by PfCRT, using alanine-scanning mutagenesis, MD simulations and drug uptake assays. [ABSTRACT FROM AUTHOR]

Malaria parasite lacks canonical pathways for amino acid biosynthesis and depends primarily on hemoglobin degradation and extracellular resources for amino acids. Interestingly, a putative gene for glutamine synthetase (GS) is retained despite glutamine being an abundant amino acid in human and mosquito hosts. Here we show Plasmodium GS has evolved as a unique type I enzyme with distinct structural and regulatory properties to adapt to the asexual niche. Methionine sulfoximine (MSO) and phosphinothricin (PPT) inhibit parasite GS activity. GS is localized to the parasite cytosol and abundantly expressed in all the life cycle stages. Parasite GS displays species-specific requirement in Plasmodium falciparum (Pf) having asparagine-rich proteome. Targeting PfGS affects asparagine levels and inhibits protein synthesis through eIF2α phosphorylation leading to parasite death. Exposure of artemisinin-resistant Pf parasites to MSO and PPT inhibits the emergence of viable parasites upon artemisinin treatment. In a study looking to examine the functional significance of glutamine synthetase (GS), the authors show that in Plasmodium, GS has evolved as a distinct type I enzyme with unique biochemical and structural features that complement the parasite niche. [ABSTRACT FROM AUTHOR]

During its asexual blood stage, P. falciparum replicates via schizogony, wherein dozens of daughter cells are formed within a single parent. The basal complex, a contractile ring that separates daughter cells, is critical for schizogony. In this study, we identify a Plasmodium basal complex protein essential for basal complex maintenance. Using multiple microscopy techniques, we demonstrate that PfPPP8 is required for uniform basal complex expansion and maintenance of its integrity. We characterize PfPPP8 as the founding member of a novel family of pseudophosphatases with homologs in other Apicomplexan parasites. By co-immunoprecipitation, we identify two additional new basal complex proteins. We characterize the unique temporal localizations of these new basal complex proteins (late-arriving) and of PfPPP8 (early-departing). In this work, we identify a novel basal complex protein, determine its specific role in segmentation, identify a new pseudophosphatase family, and establish that the P. falciparum basal complex is a dynamic structure. The authors discover a pseudophosphatase that is an essential component of the basal complex, a contractile ring required for cell division of the malaria parasite. Using a combination of genetics, biochemistry, and cell biology techniques, they demonstrate that the pseudophosphatase, PfPPP8, is critical for integrity and contraction of the basal complex. [ABSTRACT FROM AUTHOR]

Zanzibar has made significant progress toward malaria elimination, but recent stagnation requires novel approaches. We developed a highly multiplexed droplet digital PCR (ddPCR)-based amplicon sequencing method targeting 35 microhaplotypes and drug-resistance loci, and successfully sequenced 290 samples from five districts covering both main islands. Here, we elucidate fine-scale Plasmodium falciparum population structure and infer relatedness and connectivity of infections using an identity-by-descent (IBD) approach. Despite high genetic diversity, we observe pronounced fine-scale spatial and temporal parasite genetic structure. Clusters of near-clonal infections on Pemba indicate persistent local transmission with limited parasite importation, presenting an opportunity for local elimination efforts. Furthermore, we observe an admixed parasite population on Unguja and detect a substantial fraction (2.9%) of significantly related infection pairs between Zanzibar and the mainland, suggesting recent importation. Our study provides a high-resolution view of parasite genetic structure across the Zanzibar archipelago and provides actionable insights for prioritizing malaria elimination efforts. Sequencing malaria parasites from low density infections in small amounts of dried blood is important for large-scale genomic surveillance. Here, the authors develop and validate a highly multiplexed droplet digital PCR-based amplicon deep sequencing assay and apply it to data from Zanzibar, Tanzania. [ABSTRACT FROM AUTHOR]

Malaria results in more than 500,000 deaths per year and the causative Plasmodium parasites continue to develop resistance to all known agents, including different antimalarial combinations. The class XIV myosin motor PfMyoA is part of a core macromolecular complex called the glideosome, essential for Plasmodium parasite mobility and therefore an attractive drug target. Here, we characterize the interaction of a small molecule (KNX-002) with PfMyoA. KNX-002 inhibits PfMyoA ATPase activity in vitro and blocks asexual blood stage growth of merozoites, one of three motile Plasmodium life-cycle stages. Combining biochemical assays and X-ray crystallography, we demonstrate that KNX-002 inhibits PfMyoA using a previously undescribed binding mode, sequestering it in a post-rigor state detached from actin. KNX-002 binding prevents efficient ATP hydrolysis and priming of the lever arm, thus inhibiting motor activity. This small-molecule inhibitor of PfMyoA paves the way for the development of alternative antimalarial treatments. Myosin A (PfMyoA) is essential for the pathogenesis of Plasmodium falciparum, the causative agent of malaria. Here we decipher the mechanism by which a small molecule inhibitor (KNX-002) of PfMyoA impedes its motor activity. [ABSTRACT FROM AUTHOR]

In vitro evolution of drug resistance is a powerful approach for identifying antimalarial targets, however, key obstacles to eliciting resistance are the parasite inoculum size and mutation rate. Here we sought to increase parasite genetic diversity to potentiate resistance selections by editing catalytic residues of Plasmodium falciparum DNA polymerase δ. Mutation accumulation assays reveal a ~5–8 fold elevation in the mutation rate, with an increase of 13–28 fold in drug-pressured lines. Upon challenge with the spiroindolone PfATP4-inhibitor KAE609, high-level resistance is obtained more rapidly and at lower inocula than wild-type parasites. Selections also yield mutants with resistance to an "irresistible" compound, MMV665794 that failed to yield resistance with other strains. We validate mutations in a previously uncharacterised gene, PF3D7_1359900, which we term quinoxaline resistance protein (QRP1), as causal for resistance to MMV665794 and a panel of quinoxaline analogues. The increased genetic repertoire available to this "mutator" parasite can be leveraged to drive P. falciparum resistome discovery. The ability to evolve Plasmodium drug resistance in vitro is challenging and time consuming. Here, Kümpornsin et al. generated a Plasmodium falciparum parasite line with an elevated mutation rate by impairing the proof-reading activity of DNA polymerase, which results in a higher mutation rate, quick resistance development, and a lower inoculum than wild type to support the identification of new antimalarial targets and understand drug resistance mechanisms. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum causes the most severe malaria in humans. Immunoglobulin M (IgM) serves as the first line of humoral defense against infection and potently activates the complement pathway to facilitate P. falciparum clearance. A number of P. falciparum proteins bind IgM, leading to immune evasion and severe disease. However, the underlying molecular mechanisms remain unknown. Here, using high-resolution cryo-electron microscopy, we delineate how P. falciparum proteins VAR2CSA, TM284VAR1, DBLMSP, and DBLMSP2 target IgM. Each protein binds IgM in a different manner, and together they present a variety of Duffy-binding-like domain-IgM interaction modes. We further show that these proteins interfere directly with IgM-mediated complement activation in vitro, with VAR2CSA exhibiting the most potent inhibitory effect. These results underscore the importance of IgM for human adaptation of P. falciparum and provide critical insights into its immune evasion mechanism. Malaria parasites use various molecular tactics to hijack IgM antibodies and escape the human immune system. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum causes the most severe form of malaria in humans. The protozoan parasite develops within erythrocytes to mature schizonts, that contain more than 16 merozoites, which egress and invade fresh erythrocytes. The aspartic protease plasmepsin X (PMX), processes proteins and proteases essential for merozoite egress from the schizont and invasion of the host erythrocyte, including the leading vaccine candidate PfRh5. PfRh5 is anchored to the merozoite surface through a 5-membered complex (PCRCR), consisting of Plasmodium thrombospondin-related apical merozoite protein, cysteine-rich small secreted protein, Rh5-interacting protein and cysteine-rich protective antigen. Here, we show that PCRCR is processed by PMX in micronemes to remove the N-terminal prodomain of PhRh5 and this activates the function of the complex unmasking a form that can bind basigin on the erythrocyte membrane and mediate merozoite invasion. The ability to activate PCRCR at a specific time in merozoite invasion most likely masks potential deleterious effects of its function until they are required. These results provide an important understanding of the essential role of PMX and the fine regulation of PCRCR function in P. falciparum biology. Plasmodium falciparum is known to secrete an aspartic protease called plasmepsin X. Here, Triglia et al present a characterisation of plasmepsin X function in the context of erythrocyte invasion and its regulation of PCRCR, a complex that anchors the leading vaccine candidate PfRh5 to the parasite surface. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum causes the most severe form of malaria in humans. The protozoan parasite develops within erythrocytes to mature schizonts, that contain more than 16 merozoites, which egress and invade fresh erythrocytes. The aspartic protease plasmepsin X (PMX), processes proteins and proteases essential for merozoite egress from the schizont and invasion of the host erythrocyte, including the leading vaccine candidate PfRh5. PfRh5 is anchored to the merozoite surface through a 5-membered complex (PCRCR), consisting of Plasmodium thrombospondin-related apical merozoite protein, cysteine-rich small secreted protein, Rh5-interacting protein and cysteine-rich protective antigen. Here, we show that PCRCR is processed by PMX in micronemes to remove the N-terminal prodomain of PhRh5 and this activates the function of the complex unmasking a form that can bind basigin on the erythrocyte membrane and mediate merozoite invasion. The ability to activate PCRCR at a specific time in merozoite invasion most likely masks potential deleterious effects of its function until they are required. These results provide an important understanding of the essential role of PMX and the fine regulation of PCRCR function in P. falciparum biology. Plasmodium falciparum is known to secrete an aspartic protease called plasmepsin X. Here, Triglia et al present a characterisation of plasmepsin X function in the context of erythrocyte invasion and its regulation of PCRCR, a complex that anchors the leading vaccine candidate PfRh5 to the parasite surface. [ABSTRACT FROM AUTHOR]

Malaria parasites like Plasmodium falciparum multiply in red blood cells (RBC), which are cleared from the bloodstream by the spleen when their deformability is altered. Drug-induced stiffening of Plasmodium falciparum-infected RBC should therefore induce their elimination from the bloodstream. Here, based on this original mechanical approach, we identify safe drugs with strong potential to block the malaria transmission. By screening 13 555 compounds with spleen-mimetic microfilters, we identified 82 that target circulating transmissible form of P. falciparum. NITD609, an orally administered PfATPase inhibitor with known effects on P. falciparum, killed and stiffened transmission stages in vitro at nanomolar concentrations. Short exposures to TD-6450, an orally-administered NS5A hepatitis C virus inhibitor, stiffened transmission parasite stages and killed asexual stages in vitro at high nanomolar concentrations. A Phase 1 study in humans with a primary safety outcome and a secondary pharmacokinetics outcome (https://clinicaltrials.gov, ID: NCT02022306) showed no severe adverse events either with single or multiple doses. Pharmacokinetic modelling showed that these concentrations can be reached in the plasma of subjects receiving short courses of TD-6450. This physiologically relevant screen identified multiple mechanisms of action, and safe drugs with strong potential as malaria transmission-blocking agents which could be rapidly tested in clinical trials. Authors propose their splenic mimetic filtration method, microsphiltration, and utilise this approach in a drug-screen, to identify compounds that induce a stiffening effect on Plasmodium falciparum-infected erythrocytes. They proceed to assess safety and tolerability of one identified compound in a phase I clinical trial. [ABSTRACT FROM AUTHOR]

By lacking de novo purine biosynthesis enzymes, Plasmodium falciparum requires purine nucleoside uptake from host cells. The indispensable nucleoside transporter ENT1 of P. falciparum facilitates nucleoside uptake in the asexual blood stage. Specific inhibitors of PfENT1 prevent the proliferation of P. falciparum at submicromolar concentrations. However, the substrate recognition and inhibitory mechanism of PfENT1 are still elusive. Here, we report cryo-EM structures of PfENT1 in apo, inosine-bound, and inhibitor-bound states. Together with in vitro binding and uptake assays, we identify that inosine is the primary substrate of PfENT1 and that the inosine-binding site is located in the central cavity of PfENT1. The endofacial inhibitor GSK4 occupies the orthosteric site of PfENT1 and explores the allosteric site to block the conformational change of PfENT1. Furthermore, we propose a general "rocker switch" alternating access cycle for ENT transporters. Understanding the substrate recognition and inhibitory mechanisms of PfENT1 will greatly facilitate future efforts in the rational design of antimalarial drugs. PfENT1 is a promising antimalarial drug target. Here, authors report cryo-EM structures of PfENT1 that, together with biochemical work, suggests PfENT1 is an inosine transporter and describe the inhibitory mechanism of the endofacial inhibitor, GSK4. [ABSTRACT FROM AUTHOR]

Identifying how small molecules act to kill malaria parasites can lead to new "chemically validated" targets. By pressuring Plasmodium falciparum asexual blood stage parasites with three novel structurally-unrelated antimalarial compounds (MMV665924, MMV019719 and MMV897615), and performing whole-genome sequence analysis on resistant parasite lines, we identify multiple mutations in the P. falciparum acyl-CoA synthetase (ACS) genes PfACS10 (PF3D7_0525100, M300I, A268D/V, F427L) and PfACS11 (PF3D7_1238800, F387V, D648Y, and E668K). Allelic replacement and thermal proteome profiling validates PfACS10 as a target of these compounds. We demonstrate that this protein is essential for parasite growth by conditional knockdown and observe increased compound susceptibility upon reduced expression. Inhibition of PfACS10 leads to a reduction in triacylglycerols and a buildup of its lipid precursors, providing key insights into its function. Analysis of the PfACS11 gene and its mutations point to a role in mediating resistance via decreased protein stability. Drug resistance to current antimalarials is rising and new drugs and targets are urgently needed. Here the authors identify Plasmodium falciparum acyl-CoA synthetase 10 as a new target whose inhibition leads to a decrease in triacylglycerols. [ABSTRACT FROM AUTHOR]

Microtubules are a ubiquitous eukaryotic cytoskeletal element typically consisting of 13 protofilaments arranged in a hollow cylinder. This arrangement is considered the canonical form and is adopted by most organisms, with rare exceptions. Here, we use in situ electron cryo-tomography and subvolume averaging to analyse the changing microtubule cytoskeleton of Plasmodium falciparum, the causative agent of malaria, throughout its life cycle. Unexpectedly, different parasite forms have distinct microtubule structures coordinated by unique organising centres. In merozoites, the most widely studied form, we observe canonical microtubules. In migrating mosquito forms, the 13 protofilament structure is further reinforced by interrupted luminal helices. Surprisingly, gametocytes contain a wide distribution of microtubule structures ranging from 13 to 18 protofilaments, doublets and triplets. Such a diversity of microtubule structures has not been observed in any other organism to date and is likely evidence of a distinct role in each life cycle form. This data provides a unique view into an unusual microtubule cytoskeleton of a relevant human pathogen. Microtubules are a ubiquitous eukaryotic cytoskeletal element typically consisting of 13 protofilaments arranged in a hollow cylinder. Using CryoEM and subvolume averaging, Ferreira and Pražák et al. show that Plasmodium does not adhere to a single microtubule structure. Instead, the cytoskeleton changes substantially to produce a unique, fit for purpose structure and organisation at each stage of its life cycle. [ABSTRACT FROM AUTHOR]

The malaria parasite Plasmodium falciparum causes substantial human mortality, primarily in equatorial Africa. Enriched in affected African populations, the B*53 variant of HLA-B, a cell surface protein that presents peptide antigens to cytotoxic lymphocytes, confers protection against severe malaria. Gorilla, chimpanzee, and bonobo are humans' closest living relatives. These African apes have HLA-B orthologs and are infected by parasites in the same subgenus (Laverania) as P. falciparum, but the consequences of these infections are unclear. Laverania parasites infect bonobos (Pan paniscus) at only one (TL2) of many sites sampled across their range. TL2 spans the Lomami River and has genetically divergent subpopulations of bonobos on each side. Papa-B, the bonobo ortholog of HLA-B, includes variants having a B*53-like (B07) peptide-binding supertype profile. Here we show that B07 Papa-B occur at high frequency in TL2 bonobos and that malaria appears to have independently selected for different B07 alleles in the two subpopulations. A variant of MHC class I is protective against severe malaria disease and enriched in affected African populations. Here, Wroblewski et al., characterise the consequences of malaria infection in wild bonobo populations showing that the presence of malaria drives a similar evolution in immune genes. [ABSTRACT FROM AUTHOR]

Understanding mechanisms of antibody synergy is important for vaccine design and antibody cocktail development. Examples of synergy between antibodies are well-documented, but the mechanisms underlying these relationships often remain poorly understood. The leading blood-stage malaria vaccine candidate, CyRPA, is essential for invasion of Plasmodium falciparum into human erythrocytes. Here we present a panel of anti-CyRPA monoclonal antibodies that strongly inhibit parasite growth in in vitro assays. Structural studies show that growth-inhibitory antibodies bind epitopes on a single face of CyRPA. We also show that pairs of non-competing inhibitory antibodies have strongly synergistic growth-inhibitory activity. These antibodies bind to neighbouring epitopes on CyRPA and form lateral, heterotypic interactions which slow antibody dissociation. We predict that such heterotypic interactions will be a feature of many immune responses. Immunogens which elicit such synergistic antibody mixtures could increase the potency of vaccine-elicited responses to provide robust and long-lived immunity against challenging disease targets. Antibodies can have synergistic effects, but mechanisms are not well understood. Here, Ragotte et al. identify three antibodies that bind neighbouring epitopes on CyRPA, a malaria vaccine candidate, and show that lateral interactions between the antibodies slow dissociation and inhibit parasite growth synergistically. [ABSTRACT FROM AUTHOR]

The global spread of drug resistance is a major obstacle to the treatment of Plasmodium falciparum malaria. The identification of drug-resistance genes is an essential step toward solving the problem of drug resistance. Here, we report functional screening as a new approach with which to identify drug-resistance genes in P. falciparum. Specifically, a high-coverage genomic library of a drug-resistant strain is directly generated in a drug-sensitive strain, and the resistance gene is then identified from this library using drug screening. In a pilot experiment using the strain Dd2, the known chloroquine-resistant gene pfcrt is identified using the developed approach, which proves our experimental concept. Furthermore, we identify multidrug-resistant transporter 7 (pfmdr7) as a novel candidate for a mefloquine-resistance gene from a field-isolated parasite; we suggest that its upregulation possibly confers the mefloquine resistance. These results show the usefulness of functional screening as means by which to identify drug-resistance genes. Plasmodium falciparum malaria is treated using artemisinin combination therapy (ACT), in which artemisinin is supplied along with partner drugs such as mefloquine, piperaquine, and lumefantrine. However, resistance has been reported in endemic regions. To identifying new effector genes involved in resistance, Iwanaga et al. develop a large scale transgenic screen with genomic libraries of resistance strains. Using this approach they provide evidence that transcriptional upregulation of pfmdr7 contributes to mefloquine resistance in a clinical isolate. [ABSTRACT FROM AUTHOR]

Defining mechanisms of pathogen immune evasion and neutralization are critical to develop potent vaccines and therapies. Merozoite Surface Protein 1 (MSP-1) is a malaria vaccine antigen and antibodies to MSP-1 are associated with protection from disease. However, MSP-1-based vaccines performed poorly in clinical trials in part due to a limited understanding of the protective antibody response to MSP-1 and of immune evasion by antigenic diversion. Antigenic diversion was identified as a mechanism wherein parasite neutralization by a MSP-1-specific rodent antibody was disrupted by MSP-1-specific non-inhibitory blocking/interfering antibodies. Here, we investigated a panel of MSP-1-specific naturally acquired human monoclonal antibodies (hmAbs). Structures of multiple hmAbs with diverse neutralizing potential in complex with MSP-1 revealed the epitope of a potent strain-transcending hmAb. This neutralizing epitope overlaps with the epitopes of high-affinity non-neutralizing hmAbs. Strikingly, the non-neutralizing hmAbs outcompete the neutralizing hmAb enabling parasite survival. These findings demonstrate the structural and mechanistic basis for a generalizable pathogen immune evasion mechanism through neutralizing and interfering human antibodies elicited by antigenic diversion, and provides insights required to develop potent and durable malaria interventions. The Plasmodium falciparum Merozoite Surface Protein 1 (MSP-1) is a prime vaccine candidate for malaria. Here, the authors structurally and functionally characterise a panel of naturally acquired MSP-1 specific antibodies to identify one with potent broadly neutralising activity and better understand immune evasion mechanisms. [ABSTRACT FROM AUTHOR]

Diverse compounds target the Plasmodium falciparum Na+ pump PfATP4, with cipargamin and (+)-SJ733 the most clinically-advanced. In a recent clinical trial for cipargamin, recrudescent parasites emerged, with most having a G358S mutation in PfATP4. Here, we show that PfATP4G358S parasites can withstand micromolar concentrations of cipargamin and (+)-SJ733, while remaining susceptible to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in Toxoplasma gondii ATP4, decrease the sensitivity of ATP4 to inhibition by cipargamin and (+)-SJ733, thereby protecting parasites from disruption of Na+ regulation. The G358S mutation reduces the affinity of PfATP4 for Na+ and is associated with an increase in the parasite's resting cytosolic [Na+]. However, no defect in parasite growth or transmissibility is observed. Our findings suggest that PfATP4 inhibitors in clinical development should be tested against PfATP4G358S parasites, and that their combination with unrelated antimalarials may mitigate against resistance development. In a recent clinical trial for oral administration of cipargamin in individuals with malaria, there was an emergence of recrudescent parasites with a G358S mutation in PfATP4. In this work, the authors investigate the effect of this mutation on the function of the ATPase, on parasite growth and susceptibility to antimalarial drugs. [ABSTRACT FROM AUTHOR]

An effective malaria vaccine remains a global health priority and vaccine immunogens which prevent transmission of the parasite will have important roles in multi-component vaccines. One of the most promising candidates for inclusion in a transmission-blocking malaria vaccine is the gamete surface protein Pfs48/45, which is essential for development of the parasite in the mosquito midgut. Indeed, antibodies which bind Pfs48/45 can prevent transmission if ingested with the parasite as part of the mosquito bloodmeal. Here we present the structure of full-length Pfs48/45, showing its three domains to form a dynamic, planar, triangular arrangement. We reveal where transmission-blocking and non-blocking antibodies bind on Pfs48/45. Finally, we demonstrate that antibodies which bind across this molecule can be transmission-blocking. These studies will guide the development of future Pfs48/45-based vaccine immunogens. Pfs48/45, a surface protein of Plasmodium falciparum, is a promising anti-malarial vaccine candidate whose structure is not entirely resolved. Here, the authors present the structure of the full-length molecule, and characterise the binding and activity of transmission blocking antibodies. [ABSTRACT FROM AUTHOR]

The sexual stage gametocytes of the malaria parasite, Plasmodium falciparum, adopt a falciform (crescent) shape driven by the assembly of a network of microtubules anchored to a cisternal inner membrane complex (IMC). Using 3D electron microscopy, we show that a non-mitotic microtubule organizing center (MTOC), embedded in the parasite's nuclear membrane, orients the endoplasmic reticulum and the nascent IMC and seeds cytoplasmic microtubules. A bundle of microtubules extends into the nuclear lumen, elongating the nuclear envelope and capturing the chromatin. Classical mitotic machinery components, including centriolar plaque proteins, Pfcentrin-1 and −4, microtubule-associated protein, End-binding protein-1, kinetochore protein, PfNDC80 and centromere-associated protein, PfCENH3, are involved in the nuclear microtubule assembly/disassembly process. Depolymerisation of the microtubules using trifluralin prevents elongation and disrupts the chromatin, centromere and kinetochore organisation. We show that the unusual non-mitotic hemispindle plays a central role in chromatin organisation, IMC positioning and subpellicular microtubule formation in gametocytes. The sexual blood stages of Plasmodium falciparum develop through five morphologically distinct stages culminating in mature crescent-shaped gametocytes that can be transmitted from the mammalian host to the mosquito vector. Here, Li et al. apply different microscopy and tomography approaches to characterize how the microtubule organizing center and cytoplasmic and nuclear microtubules are organized and oriented during these different stages in the absence of genome replication and mitosis. [ABSTRACT FROM AUTHOR]

Tryptophan C-mannosylation stabilizes proteins bearing a thrombospondin repeat (TSR) domain in metazoans. Here we show that Plasmodium falciparum expresses a DPY19 tryptophan C-mannosyltransferase in the endoplasmic reticulum and that DPY19-deficiency abolishes C-glycosylation, destabilizes members of the TRAP adhesin family and inhibits transmission to mosquitoes. Imaging P. falciparum gametogenesis in its entirety in four dimensions using lattice light-sheet microscopy reveals defects in ΔDPY19 gametocyte egress and exflagellation. While egress is diminished, ΔDPY19 microgametes still fertilize macrogametes, forming ookinetes, but these are abrogated for mosquito infection. The gametogenesis defects correspond with destabilization of MTRAP, which we show is C-mannosylated in P. falciparum, and the ookinete defect is concordant with defective CTRP secretion on the ΔDPY19 background. Genetic complementation of DPY19 restores ookinete infectivity, sporozoite production and C-mannosylation activity. Therefore, tryptophan C-mannosylation by DPY19 ensures TSR protein quality control at two lifecycle stages for successful transmission of the human malaria parasite. Here, Lopaticki et al. show that Plasmodium falciparum expresses a Dpy19 C-mannosyltransferase in the endoplasmic reticulum that glycosylates TSR domains. Functional characterization shows that PfDpy19 plays a critical role in transmission through mosquitoes as PfDpy19-deficiency abolishes C-glycosylation and destabilizes proteins relevant for gametogenesis and oocyst formation. [ABSTRACT FROM AUTHOR]

Tryptophan C-mannosylation stabilizes proteins bearing a thrombospondin repeat (TSR) domain in metazoans. Here we show that Plasmodium falciparum expresses a DPY19 tryptophan C-mannosyltransferase in the endoplasmic reticulum and that DPY19-deficiency abolishes C-glycosylation, destabilizes members of the TRAP adhesin family and inhibits transmission to mosquitoes. Imaging P. falciparum gametogenesis in its entirety in four dimensions using lattice light-sheet microscopy reveals defects in ΔDPY19 gametocyte egress and exflagellation. While egress is diminished, ΔDPY19 microgametes still fertilize macrogametes, forming ookinetes, but these are abrogated for mosquito infection. The gametogenesis defects correspond with destabilization of MTRAP, which we show is C-mannosylated in P. falciparum, and the ookinete defect is concordant with defective CTRP secretion on the ΔDPY19 background. Genetic complementation of DPY19 restores ookinete infectivity, sporozoite production and C-mannosylation activity. Therefore, tryptophan C-mannosylation by DPY19 ensures TSR protein quality control at two lifecycle stages for successful transmission of the human malaria parasite. Here, Lopaticki et al. show that Plasmodium falciparum expresses a Dpy19 C-mannosyltransferase in the endoplasmic reticulum that glycosylates TSR domains. Functional characterization shows that PfDpy19 plays a critical role in transmission through mosquitoes as PfDpy19-deficiency abolishes C-glycosylation and destabilizes proteins relevant for gametogenesis and oocyst formation. [ABSTRACT FROM AUTHOR]

The malaria parasite Plasmodium invades a host erythrocyte, multiplies within a parasitophorous vacuole (PV) and then ruptures the PV and erythrocyte membranes in a process known as egress. Both egress and invasion are controlled by effector proteins discharged from specialized secretory organelles. The aspartic protease plasmepsin X (PM X) regulates activity for many of these effectors, but it is unclear how PM X accesses its diverse substrates that reside in different organelles. PM X also autoprocesses to generate different isoforms. The function of this processing is not understood. We have mapped the self-cleavage sites and have constructed parasites with cleavage site mutations. Surprisingly, a quadruple mutant that remains full-length retains in vitro activity, is trafficked normally, and supports normal egress, invasion and parasite growth. The N-terminal half of the prodomain stays bound to the catalytic domain even after processing and is required for proper intracellular trafficking of PM X. We find that this enzyme cleaves microneme and exoneme substrates before discharge, while the rhoptry substrates that are dependent on PM X activity are cleaved after exoneme discharge into the PV. The data give insight into the temporal, spatial and biochemical control of this unusual but important aspartic protease. Egress of Plasmodium from host erythrocytes is mediated by effector proteins. Aspartic protease plasmepsin X (PM X) regulates the activity for many of these effectors, is essential for replication and is a promising drug target. Here, Mukherjee et al. map the self-cleavage sites of PM X, show that the N-terminal part of its prodomain is required for intracellular trafficking and correlate the maturation and subcellular activity of PM X in microneme, exoneme and rhoptry organelle function. [ABSTRACT FROM AUTHOR]

Tryptophan C-mannosylation stabilizes proteins bearing a thrombospondin repeat (TSR) domain in metazoans. Here we show that Plasmodium falciparum expresses a DPY19 tryptophan C-mannosyltransferase in the endoplasmic reticulum and that DPY19-deficiency abolishes C-glycosylation, destabilizes members of the TRAP adhesin family and inhibits transmission to mosquitoes. Imaging P. falciparum gametogenesis in its entirety in four dimensions using lattice light-sheet microscopy reveals defects in ΔDPY19 gametocyte egress and exflagellation. While egress is diminished, ΔDPY19 microgametes still fertilize macrogametes, forming ookinetes, but these are abrogated for mosquito infection. The gametogenesis defects correspond with destabilization of MTRAP, which we show is C-mannosylated in P. falciparum, and the ookinete defect is concordant with defective CTRP secretion on the ΔDPY19 background. Genetic complementation of DPY19 restores ookinete infectivity, sporozoite production and C-mannosylation activity. Therefore, tryptophan C-mannosylation by DPY19 ensures TSR protein quality control at two lifecycle stages for successful transmission of the human malaria parasite. Here, Lopaticki et al. show that Plasmodium falciparum expresses a Dpy19 C-mannosyltransferase in the endoplasmic reticulum that glycosylates TSR domains. Functional characterization shows that PfDpy19 plays a critical role in transmission through mosquitoes as PfDpy19-deficiency abolishes C-glycosylation and destabilizes proteins relevant for gametogenesis and oocyst formation. [ABSTRACT FROM AUTHOR]

Ring-infected erythrocytes are the predominant asexual stage in the peripheral circulation but are rarely investigated in the context of acquired immunity against Plasmodium falciparum malaria. Here we compare antibody-dependent phagocytosis of ring-infected parasite cultures in samples from a controlled human malaria infection (CHMI) study (NCT02739763). Protected volunteers did not develop clinical symptoms, maintained parasitaemia below a predefined threshold of 500 parasites/μl and were not treated until the end of the study. Antibody-dependent phagocytosis of both ring-infected and uninfected erythrocytes from parasite cultures was strongly correlated with protection. A surface proteomic analysis revealed the presence of merozoite proteins including erythrocyte binding antigen-175 and −140 on ring-infected and uninfected erythrocytes, providing an additional antibody-mediated protective mechanism for their activity beyond invasion-inhibition. Competition phagocytosis assays support the hypothesis that merozoite antigens are the key mediators of this functional activity. Targeting ring-stage parasites may contribute to the control of parasitaemia and prevention of clinical malaria. Here the authors show that antibody-dependent phagocytosis of ring-stage P. falciparum parasites is mediated by merozoite antigens and is a strong predictor of protection following challenge in a controlled human malaria infection study in semi-immune Kenyan adults. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum has developed extensive mechanisms to evade host immune clearance. Currently, most of our understanding is based on in vitro studies of individual parasite variant surface antigens and how this relates to the processes in vivo is not well-understood. Here, we have used a humanized mouse model to identify parasite factors important for in vivo growth. We show that upregulation of the specific PfEMP1, VAR2CSA, provides the parasite with protection from macrophage phagocytosis and clearance in the humanized mice. Furthermore, parasites adapted to thrive in the humanized mice show reduced NK cell-mediated killing through interaction with the immune inhibitory receptor, LILRB1. Taken together, these findings reveal new insights into the molecular and cellular mechanisms that the parasite utilizes to coordinate immune escape in vivo. Identification and targeting of these specific parasite variant surface antigens crucial for immune evasion provides a unique approach for therapy. During the erythrocyte (RBC) stage of P. falciparum infection variant surface antigens (VSAs) such as PfEMP1s and RIFINs expressed on RBCs are important for infection and evasion of host innate immune system. Here, Chew et al. use a NSG mouse model, which is deficient in B, T and NK cells but retains macrophages, to show that PfEMP1 surface expression is required for in vivo adaptation as well as in vitro evasion of macrophage phagocytosis. [ABSTRACT FROM AUTHOR]

The cause of malaria transmission has been known for over a century but it is still unclear whether entomological measures are sufficiently reliable to inform policy decisions in human health. Decision-making on the effectiveness of new insecticide-treated nets (ITNs) and the indoor residual spraying of insecticide (IRS) have been based on epidemiological data, typically collected in cluster-randomised control trials. The number of these trials that can be conducted is limited. Here we use a systematic review to highlight that efficacy estimates of the same intervention may vary substantially between trials. Analyses indicate that mosquito data collected in experimental hut trials can be used to parameterize mechanistic models for Plasmodium falciparum malaria and reliably predict the epidemiological efficacy of quick-acting, neuro-acting ITNs and IRS. Results suggest that for certain types of ITNs and IRS using this framework instead of clinical endpoints could support policy and expedite the widespread use of novel technologies. Estimating the effectiveness of malaria vector control interventions has typically relied on resource-intensive cluster randomised trials. Here, the authors estimate changes in malaria prevalence using entomological data from experimental hut trials, which may provide an alternative route to approval of interventions in some situations. [ABSTRACT FROM AUTHOR]

Controlled human malaria infection (CHMI) has supported Plasmodium falciparum (Pf) malaria vaccine development by providing preliminary estimates of vaccine efficacy (VE). Because CHMIs generally use Pf strains similar to vaccine strains, VE against antigenically heterogeneous Pf in the field has been required to establish VE. We increased the stringency of CHMI by selecting a Brazilian isolate, Pf7G8, which is genetically distant from the West African parasite (PfNF54) in our PfSPZ vaccines. Using two regimens to identically immunize US and Malian adults, VE over 24 weeks in the field was as good as or better than VE against CHMI at 24 weeks in the US. To explain this finding, here we quantify differences in the genome, proteome, and predicted CD8 T cell epitopes of PfNF54 relative to 704 Pf isolates from Africa and Pf7G8. We show that Pf7G8 is more distant from PfNF54 than any African isolates tested. We propose VE against Pf7G8 CHMI for providing pivotal data for malaria vaccine licensure for travelers to Africa, and potentially for endemic populations, because the genetic distance of Pf7G8 from the Pf vaccine strain makes it a stringent surrogate for Pf parasites in Africa. Here the authors show that controlled human malaria infection with a Brazilian parasite highly divergent from vaccine and West African field strains can provide estimates of vaccine efficacy in Mali, and could replace field testing, streamlining vaccine development. [ABSTRACT FROM AUTHOR]

Genetically identical cells are known to exhibit differential phenotypes in the same environmental conditions. These phenotypic variants are linked to transcriptional stochasticity and have been shown to contribute towards adaptive flexibility of a wide range of unicellular organisms. Here, we investigate transcriptional heterogeneity and stochastic gene expression in Plasmodium falciparum by performing the quasilinear multiple annealing and looping based amplification cycles (MALBAC) based amplification and single cell RNA sequencing of blood stage schizonts. Our data reveals significant transcriptional variations in the schizont stage with a distinct group of highly variable invasion gene transcripts being identified. Moreover, the data reflects several diversification processes including putative developmental "checkpoint"; transcriptomically distinct parasite sub-populations and transcriptional switches in variable gene families (var, rifin, phist). Most of these features of transcriptional variability are preserved in isogenic parasite cell populations (albeit with a lesser amplitude) suggesting a role of epigenetic factors in cell-to-cell transcriptional variations in human malaria parasites. Lastly, we apply quantitative RT-PCR and RNA-FISH approach and confirm stochastic expression of key invasion genes, such as, msp1, msp3, msp7, eba181 and ama1 which represent prime candidates for invasion-blocking vaccines. Genetically identical cells can be phenotypically diverse to allow adaptive flexibility in a given environment. This phenotypic diversity is driven by epigenetic and transcriptional variability. Here, Tripathi et al. perform scRNA-seq of isogenic and non-isogenic Plasmodium falciparum schizont populations to explore transcriptional heterogeneity and stochastic gene expression during the course of development. [ABSTRACT FROM AUTHOR]

Drug resistance and a dire lack of transmission-blocking antimalarials hamper malaria elimination. Here, we present the pantothenamide MMV693183 as a first-in-class acetyl-CoA synthetase (AcAS) inhibitor to enter preclinical development. Our studies demonstrate attractive drug-like properties and in vivo efficacy in a humanized mouse model of Plasmodium falciparum infection. The compound shows single digit nanomolar in vitro activity against P. falciparum and P. vivax clinical isolates, and potently blocks P. falciparum transmission to Anopheles mosquitoes. Genetic and biochemical studies identify AcAS as the target of the MMV693183-derived antimetabolite, CoA-MMV693183. Pharmacokinetic-pharmacodynamic modelling predict that a single 30 mg oral dose is sufficient to cure a malaria infection in humans. Toxicology studies in rats indicate a > 30-fold safety margin in relation to the predicted human efficacious exposure. In conclusion, MMV693183 represents a promising candidate for further (pre)clinical development with a novel mode of action for treatment of malaria and blocking transmission. Here, de Vries et al. perform a pre-clinical characterization of the antimalarial compound MMV693183: the compound targets acetyl-CoA synthetase, has efficacy in humanized mice against Plasmodium falciparum infection, blocks transmission to mosquito vectors, is safe in rats, and pharmacokinetic-pharmacodynamic modeling informs about a potential oral human dosing regimen. [ABSTRACT FROM AUTHOR]

The RAP (RNA-binding domain abundant in Apicomplexans) protein family has been identified in various organisms. Despite expansion of this protein family in apicomplexan parasites, their main biological functions remain unknown. In this study, we use inducible knockdown studies in the human malaria parasite, Plasmodium falciparum, to show that two RAP proteins, PF3D7_0105200 (PfRAP01) and PF3D7_1470600 (PfRAP21), are essential for parasite survival and localize to the mitochondrion. Using transcriptomics, metabolomics, and proteomics profiling experiments, we further demonstrate that these RAP proteins are involved in mitochondrial RNA metabolism. Using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (eCLIP-seq), we validate that PfRAP01 and PfRAP21 are true RNA-binding proteins and interact specifically with mitochondrial rRNAs. Finally, mitochondrial enrichment experiments followed by deep sequencing of small RNAs demonstrate that PfRAP21 controls mitochondrial rRNA expression. Collectively, our results establish the role of these RAP proteins in mitoribosome activity and contribute to further understanding this protein family in malaria parasites. The function of RNA-binding domain abundant in Apicomplexans (RAP) protein family members is largely unknown. Here, using high-throughput functional genomics, including metabolomics, Hollin et al. characterize two RAP proteins that are essential for Plasmodium falciparum survival and control mitochondrial rRNAs. [ABSTRACT FROM AUTHOR]

Understanding mechanisms of antibody synergy is important for vaccine design and antibody cocktail development. Examples of synergy between antibodies are well-documented, but the mechanisms underlying these relationships often remain poorly understood. The leading blood-stage malaria vaccine candidate, CyRPA, is essential for invasion of Plasmodium falciparum into human erythrocytes. Here we present a panel of anti-CyRPA monoclonal antibodies that strongly inhibit parasite growth in in vitro assays. Structural studies show that growth-inhibitory antibodies bind epitopes on a single face of CyRPA. We also show that pairs of non-competing inhibitory antibodies have strongly synergistic growth-inhibitory activity. These antibodies bind to neighbouring epitopes on CyRPA and form lateral, heterotypic interactions which slow antibody dissociation. We predict that such heterotypic interactions will be a feature of many immune responses. Immunogens which elicit such synergistic antibody mixtures could increase the potency of vaccine-elicited responses to provide robust and long-lived immunity against challenging disease targets. Antibodies can have synergistic effects, but mechanisms are not well understood. Here, Ragotte et al. identify three antibodies that bind neighbouring epitopes on CyRPA, a malaria vaccine candidate, and show that lateral interactions between the antibodies slow dissociation and inhibit parasite growth synergistically. [ABSTRACT FROM AUTHOR]