Solution structure and pressure response of thioredoxin-1 of Plasmodium falciparum.

We present here the solution structures of the protein thioredoxin-1 from Plasmodium falciparum (PfTrx-1), in its reduced and oxidized forms. They were determined by high-resolution NMR spectroscopy at 293 K on uniformly 13C-, 15N-enriched, matched samples allowing to identification of even small structural differences. PfTrx-1 shows an α/ß-fold with a mixed five-stranded ß-sheet that is sandwiched between 4 helices in a ß1 α1 ß2 α2 ß3 α3 ß4 ß5 α4 topology. The redox process of the CGPC motif leads to significant structural changes accompanied by larger chemical shift changes from residue Phe25 to Ile36, Thr70 to Thr74, and Leu88 to Asn91. By high-field high-pressure NMR spectroscopy, rare conformational states can be identified that potentially are functionally important and can be used for targeted drug development. We performed these experiments in the pressure range from 0.1 MPa to 200 MPa. The mean combined, random-coil corrected B1* values of reduced and oxidized thioredoxin are quite similar with -0.145 and -0.114 ppm GPa-1, respectively. The mean combined, random-coil corrected B2* values in the reduced and oxidized form are 0.179 and 0.119 ppm GPa-2, respectively. The mean ratios of the pressure coefficients B2/B1 are -0.484 and -0.831 GPa-1 in the reduced and oxidized form respectively. They differ at some points in the structure after the formation of the disulfide bond between C30 and C33. The thermodynamical description of the pressure dependence of chemical shifts requires the assumption of at least three coexisting conformational states of PfTrx-1. These three conformational states were identified in the reduced as well as in the oxidized form of the protein, therefore, they represent sub-states of the two main oxidation states of PfTrx-1.

Structure-guided design of VAR2CSA-based immunogens and a cocktail strategy for a placental malaria vaccine.

Placental accumulation of Plasmodium falciparum infected erythrocytes results in maternal anemia, low birth weight, and pregnancy loss. The parasite protein VAR2CSA facilitates the accumulation of infected erythrocytes in the placenta through interaction with the host receptor chondroitin sulfate A (CSA). Antibodies that prevent the VAR2CSA-CSA interaction correlate with protection from placental malaria, and VAR2CSA is a high-priority placental malaria vaccine antigen. Here, structure-guided design leveraging the full-length structures of VAR2CSA produced a stable immunogen that retains the critical conserved functional elements of VAR2CSA. The design expressed with a six-fold greater yield than the full-length protein and elicited antibodies that prevent adhesion of infected erythrocytes to CSA. The reduced size and adaptability of the designed immunogen enable efficient production of multiple variants of VAR2CSA for use in a cocktail vaccination strategy to increase the breadth of protection. These designs form strong foundations for the development of potent broadly protective placental malaria vaccines.

A pH Fingerprint Assay to Identify Inhibitors of Multiple Validated and Potential Antimalarial Drug Targets.

New drugs with novel modes of action are needed to safeguard malaria treatment. In recent years, millions of compounds have been tested for their ability to inhibit the growth of asexual blood-stage Plasmodium falciparum parasites, resulting in the identification of thousands of compounds with antiplasmodial activity. Determining the mechanisms of action of antiplasmodial compounds informs their further development, but remains challenging. A relatively high proportion of compounds identified as killing asexual blood-stage parasites show evidence of targeting the parasite's plasma membrane Na+-extruding, H+-importing pump, PfATP4. Inhibitors of PfATP4 give rise to characteristic changes in the parasite's internal [Na+] and pH. Here, we designed a "pH fingerprint" assay that robustly identifies PfATP4 inhibitors while simultaneously allowing the detection of (and discrimination between) inhibitors of the lactate:H+ transporter PfFNT, which is a validated antimalarial drug target, and the V-type H+ ATPase, which was suggested as a possible target of the clinical candidate ZY19489. In our pH fingerprint assays and subsequent secondary assays, ZY19489 did not show evidence for the inhibition of pH regulation by the V-type H+ ATPase, suggesting that it has a different mode of action in the parasite. The pH fingerprint assay also has the potential to identify protonophores, inhibitors of the acid-loading Cl- transporter(s) (for which the molecular identity(ies) remain elusive), and compounds that act through inhibition of either the glucose transporter PfHT or glycolysis. The pH fingerprint assay therefore provides an efficient starting point to match a proportion of antiplasmodial compounds with their mechanisms of action.

Plasmodium falciparum heat shock proteins as antimalarial drug targets: An update.

Global efforts to eradicate malaria are threatened by multiple factors, particularly the emergence of antimalarial drug resistant strains of Plasmodium falciparum. Heat shock proteins (HSPs), particularly P. falciparum HSPs (PfHSPs), represent promising drug targets due to their essential roles in parasite survival and virulence across the various life cycle stages. Despite structural similarities between human and malarial HSPs posing challenges, there is substantial evidence for subtle differences that could be exploited for selective drug targeting. This review provides an update on the potential of targeting various PfHSP families (particularly PfHSP40, PfHSP70, and PfHSP90) and their interactions within PfHSP complexes as a strategy to develop new antimalarial drugs. In addition, the need for a deeper understanding of the role of HSP complexes at the host-parasite interface is highlighted, especially heterologous partnerships between human and malarial HSPs, as this opens novel opportunities for targeting protein-protein interactions crucial for malaria parasite survival and pathogenesis.

Cryogenic electron tomography reveals novel structures in the apical complex of Plasmodium falciparum.

Intracellular infectious agents, like the malaria parasite, Plasmodium falciparum, face the daunting challenge of how to invade a host cell. This problem may be even harder when the host cell in question is the enucleated red blood cell, which lacks the host machinery co-opted by many pathogens for internalization. Evolution has provided P. falciparum and related single-celled parasites within the phylum Apicomplexa with a collection of organelles at their apical end that mediate invasion. This apical complex includes at least two sets of secretory organelles, micronemes and rhoptries, and several structural features like apical rings and a putative pore through which proteins may be introduced into the host cell during invasion. We perform cryogenic electron tomography (cryo-ET) equipped with Volta Phase Plate on isolated and vitrified merozoites to visualize the apical machinery. Through tomographic reconstruction of cellular compartments, we see new details of known structures like the rhoptry tip interacting directly with a rosette resembling the recently described rhoptry secretory apparatus (RSA), or with an apical vesicle docked beneath the RSA. Subtomogram averaging reveals that the apical rings have a fixed number of repeating units, each of which is similar in overall size and shape to the units in the apical rings of tachyzoites of Toxoplasma gondii. Comparison of these polar rings in Plasmodium and Toxoplasma parasites also reveals them to have a structurally conserved assembly pattern. These results provide new insight into the essential and structurally conserved features of this remarkable machinery used by apicomplexan parasites to invade their respective host cells. IMPORTANCE: Malaria is an infectious disease caused by parasites of the genus Plasmodium and is a leading cause of morbidity and mortality globally. Upon infection, Plasmodium parasites invade and replicate in red blood cells, where they are largely protected from the immune system. To enter host cells, the parasites employ a specialized apparatus at their anterior end. In this study, advanced imaging techniques like cryogenic electron tomography (cryo-ET) and Volta Phase Plate enable unprecedented visualization of whole Plasmodium falciparum merozoites, revealing previously unknown structural details of their invasion machinery. Key findings include new insights into the structural conservation of apical rings shared between Plasmodium and its apicomplexan cousin, Toxoplasma. These discoveries shed light on the essential and conserved elements of the invasion machinery used by these pathogens. Moreover, the research provides a foundation for understanding the molecular mechanisms underlying parasite-host interactions, potentially informing strategies for combating diseases caused by apicomplexan parasites.

Plasmodium falciparum selectively degrades α-spectrin of infected erythrocytes after invasion.

Remodeling the erythrocyte membrane and skeleton by the malarial parasite Plasmodium falciparum is closely associated with intraerythrocytic development. However, the mechanisms underlying this association remain unclear. In this study, we present evidence that erythrocytic α-spectrin, but not ß-spectrin, was dynamically ubiquitinated and progressively degraded during the intraerythrocytic development of P. falciparum, from the ring to the schizont stage. We further observed an upregulated expression of P. falciparum phosphatidylinositol 3-kinase (PfPI3K) in the infected red blood cells during the intraerythrocytic development of the parasite. The data indicated that PfPI3K phosphorylated and activated erythrocytic ubiquitin-protein ligase, leading to increased α-spectrin ubiquitination and degradation during P. falciparum development. We further revealed that inhibition of the activity of PfPI3K impaired P. falciparum development in vitro and Plasmodium berghei infectivity in mice. These findings collectively unveil an important mechanism of PfPI3K-ubiquitin-mediated degradation of α-spectrin during the intraerythrocytic development of Plasmodium species. Proteins in the PfPI3K regulatory pathway are novel targets for effective treatment of severe malaria. IMPORTANCE: Plasmodium falciparum is the causative agent of severe malaria that causes millions of deaths globally. The parasite invades human red blood cells and induces a cascade of alterations in erythrocytes for development and proliferation. Remodeling the host erythrocytic cytoskeleton is a necessary process during parasitization, but its regulatory mechanisms remain to be elucidated. In this study, we observed that erythrocytic α-spectrin is selectively degraded after P. falciparum invasion, while ß-spectrin remained intact. We found that the α-spectrin chain was profoundly ubiquitinated by E3 ubiquitin ligase and degraded by the 26S proteasome. E3 ubiquitin ligase activity was regulated by P. falciparum phosphatidylinositol 3-kinase (PfPI3K) signaling. Additionally, blocking the PfPI3K-ubiquitin-proteasome pathway in P. falciparum-infected red blood cells reduced parasite proliferation and infectivity. This study deepens our understanding of the regulatory mechanisms of host and malarial parasite interactions and paves the way for the exploration of novel antimalarial drugs.

Microsatellites reveal high polymorphism and high potential for use in anti-malarial efficacy studies in areas with different transmission intensities in mainland Tanzania.

BACKGROUND: Tanzania is currently implementing therapeutic efficacy studies (TES) in areas of varying malaria transmission intensities as per the World Health Organization (WHO) recommendations. In TES, distinguishing reinfection from recrudescence is critical for the determination of anti-malarial efficacy. Recently, the WHO recommended genotyping polymorphic coding genes, merozoite surface proteins 1 and 2 (msp1 and msp2), and replacing the glutamate-rich protein (glurp) gene with one of the highly polymorphic microsatellites in Plasmodium falciparum to adjust the efficacy of antimalarials in TES. This study assessed the polymorphisms of six neutral microsatellite markers and their potential use in TES, which is routinely performed in Tanzania. METHODS: Plasmodium falciparum samples were obtained from four TES sentinel sites, Kibaha (Pwani), Mkuzi (Tanga), Mlimba (Morogoro) and Ujiji (Kigoma), between April and September 2016. Parasite genomic DNA was extracted from dried blood spots on filter papers using commercial kits. Genotyping was done using six microsatellites (Poly-α, PfPK2, TA1, C3M69, C2M34 and M2490) by capillary method, and the data were analysed to determine the extent of their polymorphisms and genetic diversity at the four sites. RESULTS: Overall, 83 (88.3%) of the 94 samples were successfully genotyped (with positive results for ≥ 50.0% of the markers), and > 50.0% of the samples (range = 47.6-59.1%) were polyclonal, with a mean multiplicity of infection (MOI) ranging from 1.68 to 1.88 among the four sites. There was high genetic diversity but limited variability among the four sites based on mean allelic richness (RS = 7.48, range = 7.27-8.03, for an adjusted minimum sample size of 18 per site) and mean expected heterozygosity (He = 0.83, range = 0.80-0.85). Cluster analysis of haplotypes using STRUCTURE, principal component analysis, and pairwise genetic differentiation (FST) did not reveal population structure or clustering of parasites according to geographic origin. Of the six markers, Poly-α was the most polymorphic, followed by C2M34, TA1 and C3M69, while M2490 was the least polymorphic. CONCLUSION: Microsatellite genotyping revealed high polyclonality and genetic diversity but no significant population structure. Poly-α, C2M34, TA1 and C3M69 were the most polymorphic markers, and Poly-α alone or with any of the other three markers could be adopted for use in TES in Tanzania.

Role for gene conversion in the evolution of cell-surface antigens of the malaria parasite Plasmodium falciparum.

While the malaria parasite Plasmodium falciparum has low average genome-wide diversity levels, likely due to its recent introduction from a gorilla-infecting ancestor (approximately 10,000 to 50,000 years ago), some genes display extremely high diversity levels. In particular, certain proteins expressed on the surface of human red blood cell-infecting merozoites (merozoite surface proteins (MSPs)) possess exactly 2 deeply diverged lineages that have seemingly not recombined. While of considerable interest, the evolutionary origin of this phenomenon remains unknown. In this study, we analysed the genetic diversity of 2 of the most variable MSPs, DBLMSP and DBLMSP2, which are paralogs (descended from an ancestral duplication). Despite thousands of available Illumina WGS datasets from malaria-endemic countries, diversity in these genes has been hard to characterise as reads containing highly diverged alleles completely fail to align to the reference genome. To solve this, we developed a pipeline leveraging genome graphs, enabling us to genotype them at high accuracy and completeness. Using our newly- resolved sequences, we found that both genes exhibit 2 deeply diverged lineages in a specific protein domain (DBL) and that one of the 2 lineages is shared across the genes. We identified clear evidence of nonallelic gene conversion between the 2 genes as the likely mechanism behind sharing, leading us to propose that gene conversion between diverged paralogs, and not recombination suppression, can generate this surprising genealogy; a model that is furthermore consistent with high diversity levels in these 2 genes despite the strong historical P. falciparum transmission bottleneck.

Selective targeting of Plasmodium falciparum Hsp90 disrupts the 26S proteasome.

The molecular chaperone heat shock protein 90 (Hsp90) has an essential but largely undefined role in maintaining proteostasis in Plasmodium falciparum, the most lethal malaria parasite. Herein, we identify BX-2819 and XL888 as potent P. falciparum (Pf)Hsp90 inhibitors. Derivatization of XL888's scaffold led to the development of Tropane 1, as a PfHsp90-selective binder with nanomolar affinity. Hsp90 inhibitors exhibit anti-Plasmodium activity against the liver, asexual blood, and early gametocyte life stages. Thermal proteome profiling was implemented to assess PfHsp90-dependent proteome stability, and the proteasome-the main site of cellular protein recycling-was enriched among proteins with perturbed stability upon PfHsp90 inhibition. Subsequent biochemical and cellular studies suggest that PfHsp90 directly promotes proteasome hydrolysis by chaperoning the active 26S complex. These findings expand our knowledge of the PfHsp90-dependent proteome and protein quality control mechanisms in these pathogenic parasites, as well as further characterize this chaperone as a potential antimalarial drug target.

Insights from structure-activity relationships and the binding mode of peptidic α-ketoamide inhibitors of the malaria drug target subtilisin-like SUB1.

Plasmodium multi-resistance, including against artemisinin, seriously threatens malaria treatment and control. Hence, new drugs are urgently needed, ideally targeting different parasitic stages, which are not yet targeted by current drugs. The SUB1 protease is involved in both hepatic and blood stages due to its essential role in the egress of parasites from host cells, and, as potential new target, it would meet the above criteria. We report here the synthesis as well as the biological and structural evaluation of substrate-based α-ketoamide SUB1 pseudopeptidic inhibitors encompassing positions P4-P2'. By individually substituting each position of the reference compound 1 (MAM-117, Ac-Ile-Thr-Ala-AlaCO-Asp-Glu (Oall)-NH2), we better characterized the structural determinants for SUB1 binding. We first identified compound 8 with IC50 values of 50 and 570 nM against Pv- and PfSUB1, respectively (about 3.5-fold higher potency compared to 1). Compound 8 inhibited P. falciparum merozoite egress in culture by 37% at 100 µM. By increasing the overall hydrophobicity of the compounds, we could improve the PfSUB1 inhibition level and antiparasitic activity, as shown with compound 40 (IC50 values of 12 and 10 nM against Pv- and PfSUB1, respectively, IC50 value of 23 µM on P. falciparum merozoite egress). We also found that 8 was highly selective towards SUB1 over three mammalian serine peptidases, supporting the promising value of this compound. Finally, several crystal 3D-structures of SUB1-inhibitor complexes, including with 8, were solved at high resolution to decipher the binding mode of these compounds.

PEXEL is a proteolytic maturation site for both exported and non-exported Plasmodium proteins.

Obligate intracellular malaria parasites dramatically remodel their erythrocyte host through effector protein export to create a niche for survival. Most exported proteins contain a pentameric Plasmodium export element (PEXEL)/host-targeting motif that is cleaved in the parasite ER by the aspartic protease Plasmepsin V (PMV). This processing event exposes a mature N terminus required for translocation into the host cell and is not known to occur in non-exported proteins. Here, we report that the non-exported parasitophorous vacuole protein UIS2 contains a bona fide PEXEL motif that is processed in the P. falciparum blood stage. While the N termini of exported proteins containing the PEXEL and immediately downstream ~10 residues are sufficient to mediate translocation into the RBC, the equivalent UIS2 N terminus does not promote the export of a reporter. Curiously, the UIS2 PEXEL contains an unusual aspartic acid at the fourth position, which constitutes the extreme N-terminal residue following PEXEL cleavage (P1', RIL↓DE). Using a series of chimeric reporter fusions, we show that Asp at P1' is permissive for PMV processing but abrogates export. Moreover, mutation of this single UIS2 residue to alanine enables export, reinforcing that the mature N terminus mediates export, not PEXEL processing per se. Prompted by this observation, we further show that PEXEL sequences in the N termini of other non-exported rhoptry proteins are also processed, suggesting that PMV may be a more general secretory maturase than previously appreciated, similar to orthologs in related apicomplexans. Our findings provide new insight into the unique N-terminal constraints that mark proteins for export.IMPORTANCEHost erythrocyte remodeling by malaria parasite-exported effector proteins is critical to parasite survival and disease pathogenesis. In the deadliest malaria parasite Plasmodium falciparum, most exported proteins undergo proteolytic maturation via recognition of the pentameric Plasmodium export element (PEXEL)/host-targeting motif by the aspartic protease Plasmepsin V, which exposes a mature N terminus that is conducive for export into the erythrocyte host cell. While PEXEL processing is considered a unique mark of exported proteins, we demonstrate that PEXEL motifs are present and processed in non-exported proteins. Importantly, we show that specific residues at the variable fourth position of the PEXEL motif inhibit export despite being permissive for processing, reinforcing that features of the mature N terminus, and not PEXEL cleavage, identify cargo for export. This opens the door to further inquiry into the nature and evolution of the PEXEL motif.

Skeleton binding protein 1 localizes to the Maurer's cleft and interacts with PfHSP70-1 and PfHSP70-x in Plasmodium falciparum gametocyte-infected erythrocytes.

Plasmodium falciparum accounts for the majority of malaria deaths, due to pathology provoked by the ability of infected erythrocytes to adhere to vascular endothelium within deep tissues. The parasite recognizes endothelium by trafficking and displaying protein ligands on the surface of asexual stage infected erythrocytes, such as members of the large family of pathogenic proteins, P. falciparum erythrocyte membrane protein 1 (PfEMP1). Parasite-encoded skeleton binding protein 1 (SBP1) plays an important role in the transport of these binding-related surface proteins, via cleft-like membranous structures termed Maurer's clefts, which are present within the cytoplasm of infected erythrocytes. Erythrocytes infected with gametocyte stages accumulate in the extravascular compartment of bone marrow; and it was suggested that their surface-expressed adhesion molecule profile and protein trafficking mechanisms might differ from those in asexual stage parasites. Protein trafficking mechanisms via Maurer's clefts have been well investigated in asexual stage parasite-infected erythrocytes; but little is known regarding the gametocyte stages. In this study, we characterized SBP1 during gametocyte maturation and demonstrated that SBP1 is expressed and localizes to dot-like Maurer's cleft structures in the cytoplasm of gametocyte-infected erythrocytes. Co-immunoprecipitation and mass spectrometry assays indicated that SBP1 interacts with the molecular chaperones PfHSP70-1 and PfHSP70-x. Localization analysis suggested that some PfHSP70-1 and/or PfHSP70-x localize in a dot-like pattern within the cytoplasm of immature gametocyte-infected erythrocytes. These findings suggest that SBP1 may interact with HSP70 chaperones in the infected erythrocyte cytoplasm during the immature gametocyte stages.

Plasmodium falciparum contains functional SCF and CRL4 ubiquitin E3 ligases, and CRL4 is critical for cell division and membrane integrity.

Protein ubiquitination is essential for cellular homeostasis and regulation of several processes, including cell division and genome integrity. Ubiquitin E3 ligases determine substrate specificity for ubiquitination, and Cullin-RING E3 ubiquitin ligases (CRLs) make the largest group among the ubiquitin E3 ligases. Although conserved and most studied in model eukaryotes, CRLs remain underappreciated in Plasmodium and related parasites. To investigate the CRLs of human malaria parasite Plasmodium falciparum, we generated parasites expressing tagged P. falciparum cullin-1 (PfCullin-1), cullin-2 (PfCullin-2), Rbx1 (PfRbx1) and Skp1 (PfSkp1). PfCullin-1 and PfCullin-2 were predominantly expressed in erythrocytic trophozoite and schizont stages, with nucleocytoplasmic localization and chromatin association, suggesting their roles in different cellular compartments and DNA-associated processes. Immunoprecipitation, in vitro protein-protein interaction, and ubiquitination assay confirmed the presence of a functional Skp1-Cullin-1-Fbox (PfSCF) complex, comprising of PfCullin-1, PfRbx1, PfSkp1, PfFBXO1, and calcyclin binding protein. Immunoprecipitation, sequence analysis, and ubiquitination assay indicated that PfCullin-2 forms a functional human CRL4-like complex (PfCRL4), consisting of PfRbx1, cleavage and polyadenylation specificity factor subunit_A and WD40 repeat proteins. PfCullin-2 knock-down at the protein level, which would hinder PfCRL4 assembly, significantly decreased asexual and sexual erythrocytic stage development. The protein levels of several pathways, including protein translation and folding, lipid biosynthesis and transport, DNA replication, and protein degradation were significantly altered upon PfCullin-2 depletion, which likely reflects association of PfCRL4 with multiple pathways. PfCullin-2-depleted schizonts had poorly delimited merozoites and internal membraned structures, suggesting a role of PfCRL4 in maintaining membrane integrity. PfCullin-2-depleted parasites had a significantly lower number of nuclei/parasite than the normal parasites, indicating a crucial role of PfCRL4 in cell division. We demonstrate the presence of functional CRLs in P. falciparum, with crucial roles for PfCRL4 in cell division and maintaining membrane integrity.

Glycophorin B-PfEMP1 interaction mediates robust rosetting in Plasmodium falciparum.

P. falciparumerythrocyte membrane protein 1 (PfEMP1) is the major parasite protein responsible for rosetting by binding to host receptors such as heparan sulfate, CR1 on RBC surface. Usually monomeric protein-carbohydrate interactions are weak [1], therefore PfEMP1 binds to plasma proteins like IgM or α2-macroglobulin that facilitate its clustering on parasitized RBC surface and augment rosetting [2,3]. We show that 3D7A expresses PfEMP1, PF3D7_0412900, and employs its CIDRγ2 domain to interact with glycophorin B on uninfected RBC to form large rosettes but more importantly even in the absence of plasma proteins. Overall, we established the role of PF3D7_0412900 in rosetting as antibodies against CIDRγ2 domain reduced rosetting and also identified its receptor, glycophorin B which could provide clue why glycophorin B null phenotype, S-s-U- RBCs prevalent in malaria endemic areas is protective against severe malaria.

Prodomain-driven enzyme dimerization: a pH-dependent autoinhibition mechanism that controls Plasmodium Sub1 activity before merozoite egress.

Malaria symptoms are associated with the asexual multiplication of Plasmodium falciparum within human red blood cells (RBCs) and fever peaks coincide with the egress of daughter merozoites following the rupture of the parasitophorous vacuole (PV) and the RBC membranes. Over the last two decades, it has emerged that the release of competent merozoites is tightly regulated by a complex cascade of events, including the unusual multi-step activation mechanism of the pivotal subtilisin-like protease 1 (Sub1) that takes place in three different cellular compartments and remains poorly understood. Following an initial auto-maturation in the endoplasmic reticulum (ER) between its pro- and catalytic domains, the Sub1 prodomain (PD) undergoes further cleavages by the parasite aspartic protease plasmepsin X (PmX) within acidic secretory organelles that ultimately lead to full Sub1 activation upon discharge into the PV. Here, we report the crystal structure of full-length P. falciparum Sub1 (PfS1FL) and demonstrate, through structural, biochemical, and biophysical studies, that the atypical Plasmodium-specific Sub1 PD directly promotes the assembly of inactive enzyme homodimers at acidic pH, whereas Sub1 is primarily monomeric at neutral pH. Our results shed new light into the finely tuned Sub1 spatiotemporal activation during secretion, explaining how PmX processing and full activation of Sub1 can occur in different cellular compartments, and uncover a robust mechanism of pH-dependent subtilisin autoinhibition that plays a key role in P. falciparum merozoites egress from infected host cells.IMPORTANCEMalaria fever spikes are due to the rupture of infected erythrocytes, allowing the egress of Plasmodium sp. merozoites and further parasite propagation. This fleeting tightly regulated event involves a cascade of enzymes, culminating with the complex activation of the subtilisin-like protease 1, Sub1. Differently than other subtilisins, Sub1 activation strictly depends upon the processing by a parasite aspartic protease within acidic merozoite secretory organelles. However, Sub1 biological activity is required in the pH neutral parasitophorous vacuole, to prime effectors involved in the rupture of the vacuole and erythrocytic membranes. Here, we show that the unusual, parasite-specific Sub1 prodomain is directly responsible for its acidic-dependent dimerization and autoinhibition, required for protein secretion, before its full activation at neutral pH in a monomeric form. pH-dependent Sub1 dimerization defines a novel, essential regulatory element involved in the finely tuned spatiotemporal activation of the egress of competent Plasmodium merozoites.

Production and Purification of Plasmodium Circumsporozoite Protein in Lactococcus lactis.

Malaria is a vector-borne disease caused by Plasmodium parasites of which Plasmodium falciparum contributed to an estimated 247 million cases worldwide in 2021 (WHO malaria report 2022). The P. falciparum Circumsporozoite protein (PfCSP) covers the surface of the sporozoite which is critical to cell invasion in the human host. PfCSP is the leading pre-erythrocytic vaccine candidate and forms the basis of the RTS'S (Mosquirix®) malaria vaccine. However, high-yield production of full-length PfCSP with proper folding has been challenging. Here, we describe expression and purification of full-length PfCSP (containing 4 NVDP and 38 NANP repeats) with proper conformation by a simple three-step procedure in the Lactococcus lactis expression system.

Comparison of extraction methods in vitro Plasmodium falciparum: A 1H NMR and LC-MS joined approach.

Malaria is a parasitic disease that remains a global concern and the subject of many studies. Metabolomics has emerged as an approach to better comprehend complex pathogens and discover possible drug targets, thus giving new insights that can aid in the development of antimalarial therapies. However, there is no standardized method to extract metabolites from in vitro Plasmodium falciparum intraerythrocytic parasites, the stage that causes malaria. Additionally, most methods are developed with either LC-MS or NMR analysis in mind, and have rarely been evaluated with both tools. In this work, three extraction methods frequently found in the literature were reproduced and samples were analyzed through both LC-MS and 1H NMR, and evaluated in order to reveal which is the most repeatable and consistent through an array of different tools, including chemometrics, peak detection and annotation. The most reliable method in this study proved to be a double extraction with methanol and methanol/water (80:20, v/v). Metabolomic studies in the field should move towards standardization of methodologies and the use of both LC-MS and 1H NMR in order to make data more comparable between studies and facilitate the achievement of biologically interpretable information.

Proteome-Wide Identification of RNA-dependent proteins and an emerging role for RNAs in Plasmodium falciparum protein complexes.

Ribonucleoprotein complexes are composed of RNA, RNA-dependent proteins (RDPs) and RNA-binding proteins (RBPs), and play fundamental roles in RNA regulation. However, in the human malaria parasite, Plasmodium falciparum, identification and characterization of these proteins are particularly limited. In this study, we use an unbiased proteome-wide approach, called R-DeeP, a method based on sucrose density gradient ultracentrifugation, to identify RDPs. Quantitative analysis by mass spectrometry identifies 898 RDPs, including 545 proteins not yet associated with RNA. Results are further validated using a combination of computational and molecular approaches. Overall, this method provides the first snapshot of the Plasmodium protein-protein interaction network in the presence and absence of RNA. R-DeeP also helps to reconstruct Plasmodium multiprotein complexes based on co-segregation and deciphers their RNA-dependence. One RDP candidate, PF3D7_0823200, is functionally characterized and validated as a true RBP. Using enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP-seq), we demonstrate that this protein interacts with various Plasmodium non-coding transcripts, including the var genes and ap2 transcription factors.

Basigin mediation of Plasmodium falciparum red blood cell invasion does not require its transmembrane domain or interaction with monocarboxylate transporter 1.

Plasmodium falciparum invasion of the red blood cell is reliant upon the essential interaction of PfRh5 with the host receptor protein basigin. Basigin exists as part of one or more multiprotein complexes, most notably through interaction with the monocarboxylate transporter MCT1. However, the potential requirement for basigin association with MCT1 and the wider role of basigin host membrane context and lateral protein associations during merozoite invasion has not been established. Using genetically manipulated in vitro derived reticulocytes, we demonstrate the ability to uncouple basigin ectodomain presentation from its transmembrane domain-mediated interactions, including with MCT1. Merozoite invasion of reticulocytes is unaffected by disruption of basigin-MCT1 interaction and by removal or replacement of the basigin transmembrane helix. Therefore, presentation of the basigin ectodomain at the red blood cell surface, independent of its native association with MCT1 or other interactions mediated by the transmembrane domain, is sufficient to facilitate merozoite invasion.

Epigenetic and transcriptional regulation of cytokine production by Plasmodium falciparum-exposed monocytes.

Plasmodium falciparum infection causes the most severe form of malaria, where excessive production of proinflammatory cytokines can drive the pathogenesis of the disease. Monocytes play key roles in host defense against malaria through cytokine production and phagocytosis; however, they are also implicated in pathogenesis through excessive proinflammatory cytokine production. Understanding the underlying molecular mechanisms that contribute to inflammatory cytokine production in P. falciparum-exposed monocytes is key towards developing better treatments. Here, we provide molecular evidence that histone 3 lysine 4 (H3K4) methylation is key for inflammatory cytokine production in P. falciparum-exposed monocytes. In an established in vitro system that mimics blood stage infection, elevated proinflammatory TNF and IL-6 cytokine production is correlated with increased mono- and tri-methylated H3K4 levels. Significantly, we demonstrate through utilizing a pharmacological inhibitor of H3K4 methylation that TNF and IL-6 expression can be suppressed in P. falciparum-exposed monocytes. This elucidated epigenetic regulatory mechanism, controlling inflammatory cytokine production, potentially provides new therapeutic options for future malaria treatment.