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Plasmodium falciparum malaria parasites invade and multiply inside red blood cells (RBCs), the most iron-rich compartment in humans. Like all cells, P. falciparum requires nutritional iron to support essential metabolic pathways, but the critical mechanisms of iron acquisition and trafficking during RBC infection have remained obscure. Parasites internalize and liberate massive amounts of heme during large-scale digestion of RBC hemoglobin within an acidic food vacuole (FV) but lack a heme oxygenase to release porphyrin-bound iron. Although most FV heme is sequestered into inert hemozoin crystals, prior studies indicate that trace heme escapes biomineralization and is susceptible to nonenzymatic degradation within the oxidizing FV environment to release labile iron. Parasites retain a homolog of divalent metal transporter 1 (DMT1), a known mammalian iron transporter, but its role in P. falciparum iron acquisition has not been tested. Our phylogenetic studies indicate that P. falciparum DMT1 (PfDMT1) retains conserved molecular features critical for metal transport. We localized this protein to the FV membrane and defined its orientation in an export-competent topology. Conditional knockdown of PfDMT1 expression is lethal to parasites, which display broad cellular defects in iron-dependent functions, including impaired apicoplast biogenesis and mitochondrial polarization. Parasites are selectively rescued from partial PfDMT1 knockdown by supplementation with exogenous iron, but not other metals. These results support a cellular paradigm whereby PfDMT1 is the molecular gatekeeper to essential iron acquisition by blood-stage malaria parasites and suggest that therapeutic targeting of PfDMT1 may be a potent antimalarial strategy., Competing Interests: Competing interests statement:The authors declare no competing interest.

Artemisinin-based combination therapy (ACT) is the mainstay of effective treatment of Plasmodium falciparum malaria. However, the long-term utility of ACTs is imperiled by widespread partial artemisinin resistance in Southeast Asia and its recent emergence in parts of East Africa. This underscores the need to identify chemotypes with new modes of action (MoAs) to circumvent resistance to ACTs. In this study, we characterized the asexual blood stage antiplasmodial activity and resistance mechanisms of LDT-623, a 4-aminoquinoline (4-AQ). We also detected LDT-623 activity against multiple stages (liver schizonts, stage IV-V gametocytes, and ookinetes) of Plasmodium's life cycle, a feature unlike other 4-AQs such as chloroquine (CQ) and piperaquine (PPQ). Using heme fractionation profiling and drug uptake studies in PfCRT-containing proteoliposomes, we observed inhibition of hemozoin formation and PfCRT-mediated transport, which constitute characteristic features of 4-AQs' MoA. We also found minimal cross-resistance to LDT-623 in a panel of mutant pfcrt or pfmdr1 lines, but not the PfCRT F145I mutant that is highly resistant to PPQ resistance yet is very unfit. No P. falciparum parasites were recovered in an in vitro resistance selection study, suggesting a high barrier for resistance to emerge. Finally, a competitive growth assay comprising >50 barcoded parasite lines with mutated resistance mediators or major drug targets found no evidence of cross-resistance. Our findings support further exploration of this promising 4-AQ., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ferreira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The natural product 9-methoxystrobilurin G (9MG) from Favolaschia spp basidiomycetes is a potent and selective antimalarial. The mechanism of action of 9MG is unknown. We induced 9MG resistance in Plasmodium falciparum 3D7 and Dd2 strains and identified mutations associated with resistance by genome sequencing. All 9MG-resistant clones possessed missense mutations in the cytochrome b (CYTB) gene, a key component of mitochondrial complex III. The mutations map to the quinol oxidation site of CYTB, which is also the target of antimalarials such as atovaquone. In a complementary approach to identify protein targets of 9MG, a photoactivatable derivative of 9MG was synthesized and applied in chemoproteomic-based target profiling. Three components of mitochondrial complex III (QCR7, QCR9, and COX15) were specifically enriched consistent with 9MG targeting CYTB and complex III function in P. falciparum . Inhibition of complex III activity by 9MG was confirmed by ubiquinone cytochrome c reductase assay using P. falciparum extract. The findings from this study may be useful for developing novel antimalarials targeting CYTB.

Inhibition of the lactate transporter PfFNT is a valid novel mode of action against malaria parasites. Current pyridine-substituted pentafluoro-3-hydroxy-pent-2-en-1-ones act as substrate analogs with submicromolar EC 50 in vitro, and >99.7% activity in mice. The recently solved structure of a PfFNT-inhibitor complex visualized the binding mode. Here, we extended the inhibitor layout by series of amine- and anilide-linked pyridine p-substituents to generate additional interactions in the cytoplasmic vestibule. Virtual docking indicated hydrogen bonding to Tyr31 and Ser102. Fluorescence cross-correlation spectroscopy yielded respectively enhanced target affinity. Strikingly, the in vitro activity increased by 1 order of magnitude to 14.8 nM at negligible cytotoxicity. While p -amine substitutions were rapidly metabolized, the more stable p -acetanilide cleared 99.7% of parasites at 4 × 50 mg kg -1 in a mouse malaria model. Future stabilization of the p-substitution against metabolism may translate the gain in in vitro potency to the in vivo situation.

Dynamic control of gene expression is critical for blood stage development of malaria parasites. Here, we used multi-omic analyses to investigate transcriptional regulation by the chromatin-associated microrchidia protein, MORC, during asexual blood stage development of the human malaria parasite Plasmodium falciparum . We show that Pf MORC (PF3D7_1468100) interacts with a suite of nuclear proteins, including APETALA2 (ApiAP2) transcription factors ( Pf AP2-G5, Pf AP2-O5, Pf AP2-I, PF3D7_0420300, PF3D7_0613800, PF3D7_1107800, and PF3D7_1239200), a DNA helicase DS60 (PF3D7_1227100), and other chromatin remodelers ( Pf CHD1 and Pf EELM2). Transcriptomic analysis of Pf MORC HA-glmS knockdown parasites revealed 163 differentially expressed genes belonging to hypervariable multigene families, along with upregulation of genes mostly involved in host cell invasion. In vivo genome-wide chromatin occupancy analysis during both trophozoite and schizont stages of development demonstrates that Pf MORC is recruited to repressed, multigene families, including the var genes in subtelomeric chromosomal regions. Collectively, we find that Pf MORC is found in chromatin complexes that play a role in the epigenetic control of asexual blood stage transcriptional regulation and chromatin organization., Competing Interests: MS, VB, IT, VS, MM, JA, CD, GP, ML, CG No competing interests declared, (© 2023, Singh, Bonnell et al.)

Malaria is a mosquito-borne infectious disease caused by unicellular eukaryotic parasites of the Plasmodium genus. Protein ubiquitination by E3 ligases is a critical post-translational modification required for various cellular processes during the lifecycle of Plasmodium parasites. However, little is known about the repertoire and function of these enzymes in Plasmodium . Here, we show that Plasmodium expresses a conserved cullin RING E3 ligase (CRL) complex that is functionally related to CRL4 in other eukaryotes. In P. falciparum asexual blood stages, a cullin-4 scaffold interacts with the RING protein RBX1, the adaptor protein DDB1, and a set of putative receptor proteins that may determine substrate specificity for ubiquitination. These receptor proteins contain WD40-repeat domains and include W D-repeat protein i mportant for g ametogenesis 1 (WIG1). This CRL4-related complex is also expressed in P. berghei gametocytes, with WIG1 being the only putative receptor detected in both the schizont and gametocyte stages. WIG1 disruption leads to a complete block in microgamete formation. Proteomic analyses indicate that WIG1 disruption alters proteostasis of ciliary proteins and components of the DNA replication machinery during gametocytogenesis. Further analysis by ultrastructure expansion microscopy (U-ExM) indicates that WIG1-dependent depletion of ciliary proteins is associated with impaired the formation of the microtubule organization centers that coordinate mitosis with axoneme formation and altered DNA replication during microgametogenesis. This work identifies a CRL4-related ubiquitin ligase in Plasmodium that is critical for the formation of microgametes by regulating proteostasis of ciliary and DNA replication proteins.IMPORTANCE Plasmodium parasites undergo fascinating lifecycles with multiple developmental steps, converting into morphologically distinct forms in both their mammalian and mosquito hosts. Protein ubiquitination by ubiquitin ligases emerges as an important post-translational modification required to control multiple developmental stages in Plasmodium . Here, we identify a cullin RING E3 ubiquitin ligase (CRL) complex expressed in the replicating asexual blood stages and in the gametocyte stages that mediate transmission to the mosquito. WIG1, a putative substrate recognition protein of this ligase complex, is essential for the maturation of microgametocytes into microgametes upon ingestion by a mosquito. More specifically, WIG1 is required for proteostasis of ciliary proteins and components of the DNA replication machinery during gametocytogenesis. This requirement is linked to DNA replication and microtubule organization center formation, both critical to the development of flagellated microgametes., Competing Interests: The authors declare no conflict of interest.

Conditional regulation is a highly beneficial system for studying the function of essential genes in Plasmodium falciparum and dimerizable Cre recombinase (DiCre) is a recently adapted conditional regulation system suitable for this purpose. In the DiCre system, two inactive fragments of Cre are reconstituted to form a functionally active enzyme in the presence of rapamycin. Different loci have been targeted to generate parasite lines that express the DiCre enzyme. Here, we have used marker-free CRISPR-Cas9 gene editing to integrate the DiCre cassette in a redundant cg6 locus. We have shown the utility of the newly generated ∆cg6DC4 parasites in mediating robust, rapid, and highly specific excision of exogenously encoded gfp sequence. The ∆cg6DC4 parasites are also capable of conditional excision of an endogenous parasite gene, PF3D7_1246000. Conditional deletion of PF3D7_1246000 did not cause any inhibition in the asexual proliferation of the parasites. Furthermore, the health and morphology of the mutant parasites were comparable to that of the control parasites in Giemsa smears. The availability of another stable DiCre parasite strain competent for conditional excision of target genes will expedite functional characterization and validation of novel drug and vaccine targets against malaria., (© 2024. The Author(s).)

Malaria is a deadly disease caused by Plasmodium parasites. While potent drugs are available in the market for malaria treatment, over the years, Plasmodium parasites have successfully developed resistance against many, if not all, front-line drugs. This poses a serious threat to global malaria eradication efforts, and the continued discovery of new drugs is necessary to tackle this debilitating disease. With recent unprecedented progress in machine learning techniques, single-cell transcriptomic in Plasmodium offers a powerful tool for identifying crucial proteins as a drug target and subsequent computational prediction of potential drugs. In this study, We have implemented a mutual-information-based feature reduction algorithm with a classification algorithm to select important proteins from transcriptomic datasets (sexual and asexual stages) for Plasmodium falciparum and then constructed the protein-protein interaction (PPI) networks of the proteins. The analysis of this PPI network revealed key proteins vital for the survival of Plasmodium falciparum. Based on the function and identification of a few strong binding sites on a couple of these key proteins, we computationally predicted a set of potential drug molecules using a deep learning-based technique. Lead drug molecules that satisfy ADMET and drug-likeliness properties are finally reported out of the generated drugs. The study offers a general computational pipeline to identify crucial proteins using scRNA-seq data sets and further development of potential new drugs., (© 2024. The Author(s).)

The methyl-d-erythritol phosphate (MEP) pathway has emerged as an interesting target in the fight against antimicrobial resistance. The pathway is essential in many human pathogens, including Plasmodium falciparum ( Pf ), but is absent in human cells. In the present study, we report on the discovery of a new chemical class targeting IspD, the third enzyme in the pathway. Exploration of the structure-activity relationship yielded inhibitors with potency in the low-nanomolar range. Moreover, we investigated the whole-cell activity, mode of inhibition, metabolic, and plasma stability of this compound class, and conducted in vivo pharmacokinetic profiling on selected compounds. Lastly, we disclosed a new mass spectrometry (MS)-based enzymatic assay for direct IspD activity determination, circumventing the need for auxiliary enzymes. In summary, we have identified a readily synthesizable compound class, demonstrating excellent activity and a promising profile, positioning it as a valuable tool compound for advancing research on IspD.

Protein arginine methyltransferases (PRMTs) play critical roles in Plasmodium falciparum , a protozoan causing the deadliest form of malaria, making them potential targets for novel antimalarial drugs. Here, we screened 11 novel PRMT inhibitors against P. falciparum asexual growth and found that onametostat, an inhibitor for type II PRMTs, exhibited strong antimalarial activity with a half-maximal inhibitory concentration (IC 50 ) value of 1.69 ± 0.04 µM. In vitro methyltransferase activities of purified PfPRMT5 were inhibited by onametostat, and a shift of IC 50 to onametostat was found in the PfPRTM5 disruptant parasite line, indicating that PfPRTM5 is the primary target of onametostat. Consistent with the function of PfPRMT5 in mediating symmetric dimethylation of histone H3R2 (H3R2me2s) and in regulating invasion-related genes, onametostat treatment led to the reduction of H3R2me2s level in P. falciparum and caused the defects on the parasite's invasion of red blood cells. This study provides a starting point for identifying specific PRMT inhibitors with the potential to serve as novel antimalarial drugs., Competing Interests: The authors declare no conflict of interest.

Introduction: Plasmodium falciparum is the most damaging malaria pathogen and brings a heavy burden to global health. Host switching and morphological changes in P. falciparum are dependent on an effective gene expression regulatory system. C5 methylation of cytosines is a common RNA modification in eukaryotes, and the NSUN family are essential m 5 C modification executors. Currently, little is known about this family in Plasmodium spp. In this study, we focus on exploring the function of PfNSUN1 protein., Methods: An efficient CRISPR/Cas9 gene editing technique was applied to construct the PfNSUN1 knockdown strain. The knockdown efficiency was confirmed by growth curves and western blot experiments. The knockdown transcriptome data was acquired to find differentially expressed genes, and target genes of PfNSUN1 protein were identified by RNA immunoprecipitation and high-throughput sequencing experiments., Results: The efficiency of PfNSUN1 protein down-regulated was about 34%. RNA-seq data revealed that differentially expressed genes were mainly down-regulated. And there were 224, 278, 556 genes that were down-regulated with more than 2-fold changes and p-adj<0.05 at ring, trophozoite and schizont stages, respectively. PfNSUN1 protein was significantly enriched on 154 target genes, including 28S ribosomal RNA and pfap2-g5 transcription factor., Discussion: PfNSUN1 is a crucial RNA post-transcriptional modification protein in P. falciparum . It plays a pivotal role in regulating gene expression and parasite growth by targeting 28S ribosomal RNA and pfap2-g5 transcription factor., Competing Interests: Author QJ is employed by Hunan Xingchen Biotechnology Company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Tang, Fan, Lu, Jiang, Cheng, Zhang, Shen and Shang.)

Most malaria rapid diagnostic tests (RDTs) detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and PfHRP3, but deletions of pfhrp2 and phfrp3 genes make parasites undetectable by RDTs. We analyzed 19,313 public whole-genome-sequenced P. falciparum field samples to understand these deletions better. Pfhrp2 deletion only occurred by chromosomal breakage with subsequent telomere healing. Pfhrp3 deletions involved loss from pfhrp3 to the telomere and showed three patterns: no other associated rearrangement with evidence of telomere healing at breakpoint (Asia; Pattern 13 - TARE1); associated with duplication of a chromosome 5 segment containing multidrug-resistant-1 gene (Asia; Pattern 13 - 5 ++ ); and most commonly, associated with duplication of a chromosome 11 segment (Americas/Africa; Pattern 13 - 11 ++ ). We confirmed a 13-11 hybrid chromosome with long-read sequencing, consistent with a translocation product arising from recombination between large interchromosomal ribosome-containing segmental duplications. Within most 13 - 11 ++ parasites, the duplicated chromosome 11 segments were identical. Across parasites, multiple distinct haplotype groupings were consistent with emergence due to clonal expansion of progeny from intrastrain meiotic recombination. Together, these observations suggest negative selection normally removes 13 - 11 ++ pfhrp3 deletions , and specific conditions are needed for their emergence and spread including low transmission, findings that can help refine surveillance strategies., Competing Interests: NH, IK, NW, SH, RC, EL, CN, DG, JJ, JP, JB No competing interests declared, (© 2024, Hathaway, Kim et al.)

Complexome profiling is an experimental approach to identify interactions by integrating native separation of protein complexes and quantitative mass spectrometry. In a typical complexome profile, thousands of proteins are detected across typically ≤100 fractions. This relatively low resolution leads to similar abundance profiles between proteins that are not necessarily interaction partners. To address this challenge, we introduce the Gaussian Interaction Profiler (GIP), a Gaussian mixture modeling-based clustering workflow that assigns protein clusters by modeling the migration profile of each cluster. Uniquely, the GIP offers a way to prioritize actual interactors over spuriously comigrating proteins. Using previously analyzed human fibroblast complexome profiles, we show good performance of the GIP compared to other state-of-the-art tools. We further demonstrate GIP utility by applying it to complexome profiles from the transmissible lifecycle stage of malaria parasites. We unveil promising novel associations for future experimental verification, including an interaction between the vaccine target Pfs47 and the hypothetical protein PF3D7&#95;0417000. Taken together, the GIP provides methodological advances that facilitate more accurate and automated detection of protein complexes, setting the stage for more varied and nuanced analyses in the field of complexome profiling. The complexome profiling data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD050751.

Malaria rapid diagnostic tests (RDTs), which detect Plasmodium falciparum (Pf)-specific histidine-rich protein-2 (HRP2), have increasing importance for the diagnosis and control of malaria, especially also in regions where routine diagnosis by microscopy is not available. HRP2-based RDTs have a similar sensitivity to expert microscopy, but their reported low specificity can lead to high false positivity rates, particularly in high-endemic areas. Despite the widespread use of RDTs, models investigating the dynamics of HRP2 clearance following Pf treatment focus rather on short-term clearance of the protein. The goal of this observational cohort study was to determine the long-term kinetic of HRP2-levels in peripheral blood after treatment of uncomplicated malaria cases with Pf mono-infection using a 3-day course of artesunate/amodiaquine. HRP2 levels were quantified at enrollment and on days 1, 2, 3, 5, 7, 12, 17, 22, and 28 post-treatment initiation. The findings reveal an unexpectedly prolonged clearance of HRP2 after parasite clearance from capillary blood. Terminal HRP2 half-life was estimated to be 9 days after parasite clearance using a pharmacokinetic two-compartmental elimination model. These results provide evidence that HRP2 clearance has generally been underestimated, as the antigen remains detectable in capillary blood for up to 28 days following successful treatment, influencing RDT-based assessment following a malaria treatment for weeks. A better understanding of the HRP2 clearance dynamics is critical for guiding the diagnosis of malaria when relying on RDTs., Importance: Detecting Plasmodium falciparum , the parasite responsible for the severest form of malaria, typically involves microscopy, polymerase chain reaction (PCR), or rapid diagnostic tests (RDTs) targeting the histidine-rich protein 2 or 3 (HRP2/3). While microscopy and PCR quickly turn negative after the infection is cleared, HRP2 remains detectable for a prolonged period. The exact duration of HRP2 persistence had not been well defined. Our study in Gabon tracked HRP2 levels over 4 weeks, resulting in a new model for antigen clearance. We discovered that a two-compartment model accurately predicts HRP2 levels, revealing an initial rapid reduction followed by a much slower elimination phase that can take several weeks. These findings are crucial for interpreting RDT results, as lingering HRP2 can lead to false positives, impacting malaria diagnosis and treatment decisions., Competing Interests: The authors declare no conflict of interest.

Non-canonical nucleotides, generated as oxidative metabolic by-products, significantly threaten the genome integrity of Plasmodium falciparum and thereby, their survival, owing to their mutagenic effects. PfHAM1, an evolutionarily conserved inosine/xanthosine triphosphate pyrophosphohydrolase, maintains nucleotide homeostasis in the malaria parasite by removing non-canonical nucleotides, although structure-function intricacies are hitherto poorly reported. Here, we report the X-ray crystal structure of PfHAM1, which revealed a homodimeric structure, additionally validated by size-exclusion chromatography-multi-angle light scattering analysis. The two monomeric units in the dimer were aligned in a parallel fashion, and critical residues associated with substrate and metal binding were identified, wherein a notable structural difference was observed in the β-sheet main frame compared to human inosine triphosphate pyrophosphatase. PfHAM1 exhibited Mg ++ -dependent pyrophosphohydrolase activity and the highest binding affinity to dITP compared to other non-canonical nucleotides as measured by isothermal titration calorimetry. Modifying the pfham1 genomic locus followed by live-cell imaging of expressed mNeonGreen-tagged PfHAM1 demonstrated its ubiquitous presence in the cytoplasm across erythrocytic stages with greater expression in trophozoites and schizonts. Interestingly, CRISPR-Cas9/DiCre recombinase-guided pfham1-null P. falciparum survived in culture under standard growth conditions, indicating its assistive role in non-canonical nucleotide clearance during intra-erythrocytic stages. This is the first comprehensive structural and functional report of PfHAM1, an atypical nucleotide-cleansing enzyme in P. falciparum., (© 2024 Federation of European Biochemical Societies.)

Malaria is a disease caused mostly by Plasmodium falciparum, affects millions of people each year. The kinases are validated targets for malaria infection. In this study, we investigate for real and hypothetical compounds that can inhibit cyclic guanosine monophosphate (CGMP)-dependent protein kinase using molecular docking via combined similarity analysis, molecular dynamics simulations, quantitative structure activity relationship (QSAR). Using Tanimoto similarity scores, ∼8.4 million compounds were screened. Compounds that have at least 70% similarity are used in further analysis. These compounds are assessed by means of docking, MMBPSA, MMGBSA and ANI&#95;LIE. Based on consensus of different free energy methods and docking we revealed two potential inhibitors that can be useful for treatment of malaria. Apart from screening of real compounds, we have also selected the 10 most plausible hypothetical compounds by performing QSAR. By QSAR proposed pharmacophores, we generated over 247 hypothetical compounds and among them 19 molecules with lower QSAR predicted IC50 values and high docking scores were selected for further analysis. We selected the top 10 inhibitor candidates and performed MD simulations for free energy calculations like the protocol applied for real compounds. According to the free energy calculations, we suggest 2 real (C 34 H 29 F 5 N 8 O 4 S and C 30 H 27 F 2 N 7 O 2 S 2 , PubChem IDs: 140564801 and 89035196, respectively) and 2 hypothetical (C 23 H 27 FN 6 O 2 S, MOL3 and C 23 H 25 FN 6 O 2 S, MOL4) compounds that can be effective inhibitors against the protein kinase of Plasmodium falciparum.Communicated by Ramaswamy H. Sarma.

A new superior bacteria complementation model was achieved for testing antifolate compounds and investigating antifolate resistance in the dihydrofolate reductase (DHFR) enzyme of the malaria parasite. Earlier models depended on the addition of trimethoprim (TMP) to chemically suppress the host Escherichia coli (Ec) DHFR function. However, incomplete suppression of EcDHFR and potential interference of antibiotics needed to maintain plasmids for complementary gene expression can complicate the interpretations. To overcome such limitations, the folA (F) and thyA (T) genes were genetically knocked out (Δ) in E. coli BL21(DE3). The resulting EcΔFΔT cells were thymidine auxotroph where thymidine supplementation or functional complementation with heterologous DHFR-thymidylate synthase (TS) is needed to restore the loss of gene functions. When tested against pyrimethamine (PYR) and its analogs designed to target Plasmodium falciparum (Pf) DHFR-TS, the 50 % inhibitory concentration values obtained from EcΔFΔT surrogates expressing wildtype (PfTM4) or double mutant (PfK1) DHFR-TS showed strong correlations to the results obtained from the standard in vitro P. falciparum growth inhibition assay. Interestingly, while TMP had little effect on the susceptibility to PYR and analogs in EcΔFΔT expressing PfDHFR-TS, it hypersensitized the chemically knockdown E. coli BL21(DE3) expressing PfTM4 DHFR-TS but desensitized the one carrying PfK1 DHFR-TS. The low intrinsic expression level of PfTM4 in E. coli BL21(DE3) by western blot analysis may explain the hypersensitive to antifolates of chemical knockdown bacteria surrogate. These results demonstrated the usefulness of EcΔFΔT surrogate as a new tool for antifolate antimalarial screening with potential application for investigation of antifolate resistance mechanism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

PF11&#95;0189 is a putative insulin degrading enzyme present in Plasmodium falciparum genome. The catalytic domain of PF11&#95;0189 is about 27 kDa. Substrate specificity study shows PF11&#95;0189 acts upon different types of proteins. The substrate specificity is found to be highest when insulin is used as a substrate. Metal dependency study shows highest dependency of PF11&#95;0189 towards zinc metal for its proteolytic activity. Chelation of zinc metal with EDTA shows complete absence of PF11&#95;0189 activity. Peptide inhibitors, P-70 and P-121 from combinatorial peptide library prepared against PF11&#95;0189 show inhibition with an IC50 value of 4.8 μM and 7.5 μM respectively. A proven natural anti-malarial peptide cyclosporin A shows complete inhibition against PF11&#95;0189 with an IC50 value of 0.75 μM suggesting PF11&#95;0189 as a potential target for peptide inhibitors. The study implicates that PF11&#95;0189 is a zinc metalloprotease involved in catalysis of insulin. The study gives a preliminary insight into the mechanism of complications arising from glucose abnormalities during severe malaria., (Copyright © 2024 Elsevier Inc. All rights reserved.)

We report the discovery of MED6-189, an analog of the kalihinol family of isocyanoterpene natural products that is effective against drug-sensitive and drug-resistant Plasmodium falciparum strains, blocking both asexual replication and sexual differentiation. In vivo studies using a humanized mouse model of malaria confirm strong efficacy of the compound in animals with no apparent hemolytic activity or toxicity. Complementary chemical, molecular, and genomics analyses revealed that MED6-189 targets the parasite apicoplast and acts by inhibiting lipid biogenesis and cellular trafficking. Genetic analyses revealed that a mutation in PfSec13 , which encodes a component of the parasite secretory machinery, reduced susceptibility to the drug. Its high potency, excellent therapeutic profile, and distinctive mode of action make MED6-189 an excellent addition to the antimalarial drug pipeline.

Aurora kinases are crucial regulators of mitotic cell cycle progression in eukaryotes. The protozoan malaria parasite Plasmodium falciparum replicates via schizogony, a specialized mode of cell division characterized by consecutive asynchronous rounds of nuclear division by closed mitosis followed by a single cytokinesis event producing dozens of daughter cells. P. falciparum encodes three Aurora-related kinases (PfARKs) that have been reported essential for parasite proliferation, but their roles in regulating schizogony have not yet been explored in great detail. Here, we engineered transgenic parasite lines expressing GFP-tagged PfARK1-3 to provide a systematic analysis of their expression timing and subcellular localization throughout schizogony as well as in the non-dividing gametocyte stages, which are essential for malaria transmission. We demonstrate that all three PfARKs display distinct and highly specific and exclusive spatiotemporal associations with the mitotic machinery. In gametocytes, PfARK3 is undetectable, and PfARK1 and PfARK2 show male-specific expression in late-stage gametocytes, consistent with their requirement for endomitosis during male gametogenesis in the mosquito vector. Our combined data suggest that PfARK1 and PfARK2 have non-overlapping roles in centriolar plaque maturation, assembly of the mitotic spindle, kinetochore-spindle attachment and chromosome segregation, while PfARK3 seems to be exquisitely involved in daughter cell cytoskeleton assembly and cytokinesis. These important new insights provide a reliable foundation for future research aiming at the functional investigation of these divergent and possibly drug-targetable Aurora-related kinases in mitotic cell division of P. falciparum and related apicomplexan parasites.IMPORTANCEMalaria parasites replicate via non-conventional modes of mitotic cell division, such as schizogony, employed by the disease-causing stages in the human blood or endomitosis during male gametogenesis in the mosquito vector. Understanding the molecular mechanisms regulating cell division in these divergent unicellular eukaryotes is not only of scientific interest but also relevant to identify potential new antimalarial drug targets. Here, we carefully examined the subcellular localization of all three Plasmodium falciparum Aurora-related kinases (ARKs), distantly related homologs of Aurora kinases that coordinate mitosis in model eukaryotes. Detailed fluorescence microscopy-based analyses revealed distinct, specific, and exclusive spatial associations for each parasite ARK with different components of the mitotic machinery and at different phases of the cell cycle during schizogony and gametocytogenesis. This comprehensive set of results closes important gaps in our fragmentary knowledge on this important group of kinases and offers a valuable source of information for future functional studies., Competing Interests: The authors declare no conflict of interest.

Malaria pathogenesis and parasite multiplication depend on the ability of Plasmodium merozoites to invade human erythrocytes. Invasion is a complex multi-step process involving multiple parasite proteins which can differ between species and has been most extensively studied in P. falciparum. However, dissecting the precise role of individual proteins has to date been limited by the availability of quantifiable phenotypic assays. In this study, we apply a new approach to assigning function to invasion proteins by using optical tweezers to directly manipulate recently egressed P. falciparum merozoites and erythrocytes and quantify the strength of attachment between them, as well as the frequency with which such attachments occur. Using a range of inhibitors, antibodies, and genetically modified strains including some generated specifically for this work, we quantitated the contribution of individual P. falciparum proteins to these merozoite-erythrocyte attachment interactions. Conditional deletion of the major P. falciparum merozoite surface protein PfMSP1, long thought to play a central role in initial attachment, had no impact on the force needed to pull merozoites and erythrocytes apart, whereas interventions that disrupted the function of several members of the EBA-175 like Antigen (PfEBA) family and Reticulocyte Binding Protein Homologue (PfRH) invasion ligand families did have a significant negative impact on attachment. Deletion of individual PfEBA and PfRH ligands reinforced the known redundancy within these families, with the deletion of some ligands impacting detachment force while others did not. By comparing over 4000 individual merozoite-erythrocyte interactions in a range of conditions and strains, we establish that the PfEBA/PfRH families play a central role in P. falciparum merozoite attachment, not the major merozoite surface protein PfMSP1., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kals et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. For the purpose of open access, the author has applied a CC BY copyright license to any Author Accepted Manuscript Version arising from this submission.)

Development of the malaria parasite, Plasmodium falciparum, is regulated by a limited number of sequence-specific transcription factors (TFs). However, the mechanisms by which these TFs recognize genome-wide binding sites is largely unknown. To address TF specificity, we investigated the binding of two TF subsets that either bind CACACA or GTGCAC DNA sequence motifs and further characterized two additional ApiAP2 TFs, PfAP2-G and PfAP2-EXP, which bind unique DNA motifs (GTAC and TGCATGCA). We also interrogated the impact of DNA sequence and chromatin context on P. falciparum TF binding by integrating high-throughput in vitro and in vivo binding assays, DNA shape predictions, epigenetic post-translational modifications, and chromatin accessibility. We found that DNA sequence context minimally impacts binding site selection for paralogous CACACA-binding TFs, while chromatin accessibility, epigenetic patterns, co-factor recruitment, and dimerization correlate with differential binding. In contrast, GTGCAC-binding TFs prefer different DNA sequence context in addition to chromatin dynamics. Finally, we determined that TFs that preferentially bind divergent DNA motifs may bind overlapping genomic regions due to low-affinity binding to other sequence motifs. Our results demonstrate that TF binding site selection relies on a combination of DNA sequence and chromatin features, thereby contributing to the complexity of P. falciparum gene regulatory mechanisms., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Malaria is a global and deadly human disease caused by the apicomplexan parasites of the genus Plasmodium. Parasite proliferation within human red blood cells (RBCs) is associated with the clinical manifestations of the disease. This asexual expansion within human RBCs begins with the invasion of RBCs by P. falciparum, which is mediated by the secretion of effectors from 2 specialized club-shaped secretory organelles in merozoite-stage parasites known as rhoptries. We investigated the function of the Rhoptry Neck Protein 11 (RON11), which contains 7 transmembrane domains and calcium-binding EF-hand domains. We generated conditional mutants of the P. falciparum RON11. Knockdown of RON11 inhibits parasite growth by preventing merozoite invasion. The loss of RON11 did not lead to any defects in processing of rhoptry proteins but instead led to a decrease in the amount of rhoptry proteins. We utilized ultrastructure expansion microscopy (U-ExM) to determine the effect of RON11 knockdown on rhoptry biogenesis. Surprisingly, in the absence of RON11, fully developed merozoites had only 1 rhoptry each. The single rhoptry in RON11-deficient merozoites were morphologically typical with a bulb and a neck oriented into the apical polar ring. Moreover, rhoptry proteins are trafficked accurately to the single rhoptry in RON11-deficient parasites. These data show that in the absence of RON11, the first rhoptry is generated during schizogony but upon the start of cytokinesis, the second rhoptry never forms. Interestingly, these single-rhoptry merozoites were able to attach to host RBCs but are unable to invade RBCs. Instead, RON11-deficient merozoites continue to engage with RBC for prolonged periods eventually resulting in echinocytosis, a result of secreting the contents from the single rhoptry into the RBC. Together, our data show that RON11 triggers the de novo biogenesis of the second rhoptry and functions in RBC invasion., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Anaguano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Glycophosphatidylinositol (GPI) anchors are the predominant glycoconjugate in Plasmodium parasites, enabling modified proteins to associate with biological membranes. GPI biosynthesis commences with donation of a mannose residue held by dolichol-phosphate at the endoplasmic reticulum membrane. In Plasmodium dolichols are derived from isoprenoid precursors synthesised in the Plasmodium apicoplast, a relict plastid organelle of prokaryotic origin. We found that treatment of Plasmodium parasites with apicoplast inhibitors decreases the synthesis of isoprenoid and GPI intermediates resulting in GPI-anchored proteins becoming untethered from their normal membrane association. Even when other isoprenoids were chemically rescued, GPI depletion led to an arrest in schizont stage parasites, which had defects in segmentation and egress. In those daughter parasites (merozoites) that did form, proteins that would normally be GPI-anchored were mislocalised, and when these merozoites were artificially released they were able to attach to but not invade new red blood cells. Our data provides further evidence for the importance of GPI biosynthesis during the asexual cycle of P. falciparum, and indicates that GPI biosynthesis, and by extension egress and invasion, is dependent on isoprenoids synthesised in the apicoplast., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Bulloch et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Novel antimalarials are urgently needed to combat rising resistance to available drugs. The imidazolopiperazine ganaplacide is a promising drug candidate, but decreased susceptibility of laboratory strains has been linked to polymorphisms in the Plasmodium falciparum cyclic amine resistance locus (PfCARL), acetyl-CoA transporter (PfACT), and UDP-galactose transporter (PfUGT). To characterize parasites causing disease in Africa, we assessed ex vivo drug susceptibilities to ganaplacide in 750 P . falciparum isolates collected in Uganda from 2017 to 2023. Drug susceptibilities were assessed using a 72-hour SYBR Green growth inhibition assay. The median IC 50 for ganaplacide was 13.8 nM, but some isolates had up to 31-fold higher IC 50 s (31/750 with IC 50 > 100 nM). To assess genotype-phenotype associations, we sequenced genes potentially mediating altered ganaplacide susceptibility in the isolates using molecular inversion probe and dideoxy sequencing methods. PfCARL was highly polymorphic, with eight mutations present in >5% of isolates. None of these eight mutations had previously been selected in laboratory strains with in vitro drug pressure and none were found to be significantly associated with decreased ganaplacide susceptibility. Mutations in PfACT and PfUGT were found in ≤5% of isolates, except for two frequent (>20%) mutations in PfACT; one mutation in PfACT (I140V) was associated with a modest decrease in susceptibility. Overall, Ugandan P. falciparum isolates were mostly highly susceptible to ganaplacide. Known resistance mediators were polymorphic, but mutations previously selected with in vitro drug pressure were not seen, and mutations identified in the Ugandan isolates were generally not associated with decreased ganaplacide susceptibility., Competing Interests: Benjamin Blasco, Maelle Duffey, and Melanie Rouillier were employed by MMV, who funded part of the study.

The human malaria parasite Plasmodium falciparum (P. falciparum) continues to pose a significant public health challenge, leading to millions of fatalities globally. Halofuginone (HF) has shown a significant anti-P. falciparum effect, suggesting its potential as a therapeutic agent for malaria treatment. In this study, we synthesized a photoaffinity labeling probe of HF to identify its direct target in P. falciparum. Our results reveal that ubiquitin carboxyl-terminal hydrolase 3 (PfUCHL3) acts as a crucial target protein of HF, which modulates parasite growth in the intraerythrocytic cycle. In particular, we discovered that HF potentially forms hydrogen bonds with the Leu10, Glu11, and Arg217 sites of PfUCHL3, thereby inducing an allosteric effect by promoting the embedding of the helix 6' region on the protein surface. Furthermore, HF disrupts the expression of multiple functional proteins mediated by PfUCHL3, specifically those that play crucial roles in amino acid biosynthesis and metabolism in P. falciparum. Taken together, this study highlights PfUCHL3 as a previously undisclosed druggable target of HF, which contributes to the development of novel anti-malarial agents in the future., (© 2024 Wiley-VCH GmbH.)

Apicomplexan parasites are the primary causative agents of many human diseases, including malaria, toxoplasmosis, and cryptosporidiosis. These opportunistic pathogens undergo complex life cycles with multiple developmental stages, wherein many key steps are regulated by phosphorylation mechanisms. The genomes of apicomplexan pathogens contain protein kinases from different groups including tyrosine kinase-like (TKL) family proteins. Although information on the role of TKL kinases in apicomplexans is quite limited, recent studies have revealed the important role of this family of proteins in apicomplexan biology. TKL kinases in these protozoan pathogens show unique organization with many novel domains thus making them attractive candidates for drug development. In this mini review, we summarize the current understanding of the role of TKL kinases in human apicomplexan pathogens' (Toxoplasma gondii, Plasmodium falciparum and Cryptosporidium parvum) biology and pathogenesis., Competing Interests: Declaration of Competing Interest None, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Vacuolar type ATPases (V-type ATPases) are highly conserved hetero-multisubunit proton pumping machineries found in all eukaryotes. They utilize ATP hydrolysis to pump protons, acidifying intracellular or extracellular compartments, and are thus crucial for various biological processes. Despite their evolutionary conservation in malaria parasites, this proton pump remains understudied. To understand the localization and biological functions of Plasmodium falciparum V-type ATPase, we employed CRISPR/Cas9 to endogenously tag the subunit A of the V 1 domain. V 1 A (PF3D7&#95;1311900) was tagged with a triple hemagglutinin epitope and the TetR-DOZI-aptamer system for conditional expression under the regulation of anhydrotetracycline. Via immunofluorescence assays, we identified that V-type ATPase is expressed throughout the intraerythrocytic developmental cycle and is mainly localized to the digestive vacuole and parasite plasma membrane. Immuno-electron microscopy further revealed that V-type ATPase is also localized on secretory organelles in merozoites. Knockdown of V 1 A led to cytosolic pH imbalance and blockage of hemoglobin digestion in the digestive vacuole, resulting in an arrest of parasite development in the trophozoite-stage and, ultimately, parasite demise. Using bafilomycin A1, a specific inhibitor of V-type ATPases, we found that the P. falciparum V-type ATPase is likely involved in parasite invasion but is not critical for ring-stage development. Further, we detected a large molecular weight complex in blue native-PAGE (∼1.0 MDa), corresponding to the total molecular weights of V 1 and V o domains. Together, we show that V-type ATPase is localized to multiple subcellular compartments in P. falciparum, and its functionality throughout the asexual cycle varies depending on the parasite developmental stages., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: PfK13 protein mutations are associated with the emergence of artemisinin resistance in Plasmodium falciparum. PfK13 protein is essential for mediating ubiquitination and controlling the PI3K/AKT pathway. Mutant PfK13 variations can interfere with substrate binding, especially with PfPI3K, which raises PfPI3K levels., Methods: DUET, DynaMut2, mCSM, iStable 2.0, I-Mutant 2.0, and MuPro were utilized to study the protein stability and protein-substrate binding was studied using HADDOCK 2.4 docking algorithm between Wild-type and mutant PfK13 with the helical and catalytic domain of PfPI3K., Results: i-Stable server analysis predicted that seven, out of the nine mutations associated with artemisinin resistance (F446I, Y493H, R539T, I543T, P553L, R561H, C580Y) reduced the protein stability. HADDOCK scores of the catalytic domain underscores the significant impact of the reported mutations on the binding affinity of the PfK13 protein. Further validation through the MM&#95;GBSA technique, the binding free energy (DDG) between the wild-type and the mutant PfK13 protein analysis revealed a loss of interactions resulting from mutations in PfK13., Conclusion: The study finding suggest that mutations in the PfK13 cause destabilization in the protein structure and affects the binding of PfPI3K. Although the findings remain preliminary and require further validation, it provides the basis for further research considering the importance of the interaction of PfK13 and PfPI3K to overcome the impact of artemisinin resistance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

Background: The malaria parasite Plasmodium falciparum replicates within red blood cells, then ruptures the cell in a process called egress in order to continue its life cycle. Egress is regulated by a proteolytic cascade involving an essential parasite subtilisin-like serine protease called SUB1. Maturation of SUB1 initiates in the parasite endoplasmic reticulum with autocatalytic cleavage of an N-terminal prodomain (p31), which initially remains non-covalently bound to the catalytic domain, p54. Further trafficking of the p31-p54 complex results in formation of a terminal p47 form of the SUB1 catalytic domain. Recent work has implicated a parasite aspartic protease, plasmepsin X (PMX), in maturation of the SUB1 p31-p54 complex through controlled cleavage of the prodomain p31., Methods: Here we use biochemical and enzymatic analysis to examine the activation of SUB1 by PMX., Results: We show that both p31 and p31-p54 are largely dimeric under the relatively acidic conditions to which they are likely exposed to PMX in the parasite. We confirm the sites within p31 that are cleaved by PMX and determine the order of cleavage. We find that cleavage by PMX results in rapid loss of the capacity of p31 to act as an inhibitor of SUB1 catalytic activity and we directly demonstrate that exposure to PMX of recombinant p31-p54 complex activates SUB1 activity., Conclusions: Our results confirm that precise, PMX-mediated cleavage of the SUB1 prodomain activates SUB1 enzyme activity., General Significance: Our findings elucidate the role of PMX in activation of SUB1, a key effector of malaria parasite egress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The malaria-causing parasite, P. falciparum, replicates through schizogony, a tightly orchestrated process where numerous daughter parasites are formed simultaneously. Proper division and segregation of one-per-cell organelles, like the mitochondrion and apicoplast, are essential, yet remain poorly understood. We developed a new reporter parasite line that allows visualization of the mitochondrion in blood and mosquito stages. Using high-resolution 3D imaging, we found that the mitochondrion orients in a cartwheel structure, prior to stepwise, non-geometric division during last-stage schizogony. Analysis of focused ion beam scanning electron microscopy data confirmed these mitochondrial division stages. Furthermore, these data allowed us to elucidate apicoplast division steps, highlighted its close association with the mitochondrion, and showed putative roles of the centriolar plaques in apicoplast segregation. These observations form the foundation for a new detailed mechanistic model of mitochondrial and apicoplast division and segregation during P. falciparum schizogony and pave the way for future studies into the proteins and protein complexes involved in organelle division and segregation., (© 2024 Verhoef et al.)

In the absence of an efficacious vaccine, chemotherapy remains crucial to prevent and treat malaria. Given its key role in haemoglobin degradation, falcilysin constitutes an attractive target. Here, we reveal the mechanism of enzymatic inhibition of falcilysin by MK-4815, an investigational new drug with potent antimalarial activity. Using X-ray crystallography, we determine two binary complexes of falcilysin in a closed state, bound with peptide substrates from the haemoglobin α and β chains respectively. An antiparallel β-sheet is formed between the substrate and enzyme, accounting for sequence-independent recognition at positions P2 and P1. In contrast, numerous contacts favor tyrosine and phenylalanine at the P1' position of the substrate. Cryo-EM studies reveal a majority of unbound falcilysin molecules adopting an open conformation. Addition of MK-4815 shifts about two-thirds of falcilysin molecules to a closed state. These structures give atomic level pictures of the proteolytic cycle, in which falcilysin interconverts between a closed state conducive to proteolysis, and an open conformation amenable to substrate diffusion and products release. MK-4815 and quinolines bind to an allosteric pocket next to a hinge region of falcilysin and hinders this dynamic transition. These data should inform the design of potent inhibitors of falcilysin to combat malaria., (© 2024. The Author(s).)

The Plasmodium falciparum mitochondrial electron transport chain (mETC) is responsible for essential metabolic pathways such as de novo pyrimidine synthesis and ATP synthesis. The mETC complex III (cytochrome bc 1 complex) is responsible for transferring electrons from ubiquinol to cytochrome c and generating a proton gradient across the inner mitochondrial membrane, which is necessary for the function of ATP synthase. Recent studies have revealed that the composition of Plasmodium falciparum complex III (PfCIII) is divergent from humans, highlighting its suitability as a target for specific inhibition. Indeed, PfCIII is the target of the clinically used anti-malarial atovaquone and of several inhibitors undergoing pre-clinical trials, yet its role in parasite biology has not been thoroughly studied. We provide evidence that the universally conserved subunit, PfRieske, and the new parasite subunit, PfC3AP2, are part of PfCIII, with the latter providing support for the prediction of its divergent composition. Using inducible depletion, we show that PfRieske, and therefore, PfCIII as a whole, is essential for asexual blood stage parasite survival, in line with previous observations. We further found that depletion of PfRieske results in gametocyte maturation defects. These phenotypes are linked to defects in mitochondrial functions upon PfRieske depletion, including increased sensitivity to mETC inhibitors in asexual stages and decreased cristae abundance alongside abnormal mitochondrial morphology in gametocytes. This is the first study that explores the direct role of the PfCIII in gametogenesis via genetic disruption, paving the way for a better understanding of the role of mETC in the complex life cycle of these important parasites and providing further support for the focus of antimalarial drug development on this pathway.

The Plasmodium falciparum life cycle includes obligate transition between a human and mosquito host. Gametocytes are responsible for transmission from the human to the mosquito vector where gamete fusion followed by meiosis occurs. To elucidate how male and female gametocytes differentiate in the absence of sex chromosomes, we perform FACS-based cell enrichment of a P. falciparum gametocyte reporter line followed by single-cell RNA-seq. In our analyses we define the transcriptional programs and predict candidate driver genes underlying male and female development, including genes from the ApiAP2 family of transcription factors. A motif-driven, gene regulatory network analysis indicates that AP2-G5 specifically modulates male development. Additionally, genes linked to the inner membrane complex, involved in morphological changes, are uniquely expressed in the female lineage. The transcriptional programs of male and female development detailed herein allow for further exploration of the evolution of sex in eukaryotes and provide targets for future development of transmission blocking therapies., (© 2024. The Author(s).)

Malaria, an ancient mosquito-borne illness caused by Plasmodium parasites, is mostly treated with Artemisinin Combination Therapy (ACT). However, Single Nucleotide Polymorphisms (SNPs) mutations in the P. falciparum Kelch 13 (PfK13) protein have been associated with artemisinin resistance (ART-R). Therefore, this study aims to generate PfK13 recombinant proteins incorporating of two specific SNPs mutations, PfK13-V494I and PfK13-N537I, and subsequently analyze their binding interactions with artemisinin (ART). The recombinant proteins of PfK13 mutations and the Wild Type (WT) variant were expressed utilizing a standard protein expression protocol with modifications and subsequently purified via IMAC and confirmed with SDS-PAGE analysis and Orbitrap tandem mass spectrometry. The binding interactions between PfK13-V494I and PfK13-N537I propeller domain proteins ART were assessed through Isothermal Titration Calorimetry (ITC) and subsequently validated using fluorescence spectrometry. The protein concentrations obtained were 0.3 mg/ml for PfK13-WT, 0.18 mg/ml for PfK13-V494I, and 0.28 mg/ml for PfK13-N537I. Results obtained for binding interaction revealed an increased fluorescence intensity in the mutants PfK13-N537I (83 a.u.) and PfK13-V494I (143 a.u.) compared to PfK13-WT (33 a.u.), indicating increased exposure of surface proteins because of the looser binding between PfK13 protein mutants with ART. This shows that the PfK13 mutations may induce alterations in the binding interaction with ART, potentially leading to reduced effectiveness of ART and ultimately contributing to ART-R. However, this study only elucidated one facet of the contributing factors that could serve as potential indicators for ART-R and further investigation should be pursued in the future to comprehensively explore this complex mechanism of ART-R., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Md. Yusuf et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Plasmodium falciparum subtilisin-like serine protease 1 (PfSUB1) is essential for egress of invasive merozoite forms of the parasite, rendering PfSUB1 an attractive antimalarial target. Here, we report studies aimed to improve drug-like properties of peptidic boronic acid PfSUB1 inhibitors including increased lipophilicity and selectivity over human proteasome (H20S). Structure-activity relationship investigations revealed that lipophilic P 3 amino acid side chains as well as N -capping groups were well tolerated in retaining PfSUB1 inhibitory potency. At the P 1 position, replacing the methyl group with a carboxyethyl substituent led to boralactone PfSUB1 inhibitors with remarkably improved selectivity over H20S. Combining lipophilic end-capping groups with the boralactone reduced the selectivity over H20S. However, compound 4c still showed >60-fold selectivity versus H20S and low nanomolar PfSUB1 inhibitory potency. Importantly, this compound inhibited the growth of a genetically modified P. falciparum line expressing reduced levels of PfSUB1 13-fold more efficiently compared to a wild-type parasite line.

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., (© 2024. The Author(s).)

Glycosylphosphatidylinositol (GPI) anchor protein modification in Plasmodium species is well known and represents the principal form of glycosylation in these organisms. The structure and biosynthesis of GPI anchors of Plasmodium spp. has been primarily studied in the asexual blood stage of Plasmodium falciparum and is known to contain the typical conserved GPI structure of EtN-P-Man3GlcN-PI. Here, we have investigated the circumsporozoite protein (CSP) for the presence of a GPI anchor. CSP is the major surface protein of Plasmodium sporozoites, the infective stage of the malaria parasite. While it is widely assumed that CSP is a GPI-anchored cell surface protein, compelling biochemical evidence for this supposition is absent. Here, we employed metabolic labeling and mass-spectrometry-based approaches to confirm the presence of a GPI anchor in CSP. Biosynthetic radiolabeling of CSP with [ 3 H]-palmitic acid and [ 3 H]-ethanolamine, with the former being base-labile and therefore ester-linked, provided strong evidence for the presence of a GPI anchor on CSP, but these data alone were not definitive. To provide further evidence, immunoprecipitated CSP was analyzed for the presence of myo-inositol (a characteristic component of GPI anchor) using strong acid hydrolysis and GC-MS for highly sensitive and quantitative detection. The single ion monitoring (SIM) method for GC-MS analysis confirmed the presence of the myo-inositol component in CSP. Taken together, these data provide confidence that the long-assumed presence of a GPI anchor on this important parasite protein is correct., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Target-based approaches have traditionally been used in the search for new anti-infective molecules. Target selection process, a critical step in Drug Discovery, identifies targets that are essential to establish or maintain the infection, tractable to be susceptible for inhibition, selective towards their human ortholog and amenable for large scale purification and high throughput screening. The work presented herein validates the Plasmodium falciparum mRNA 5' triphosphatase (PfPRT1), the first enzymatic step to cap parasite nuclear mRNAs, as a candidate target for the development of new antimalarial compounds. mRNA capping is essential to maintain the integrity and stability of the messengers, allowing their translation. PfPRT1 has been identified as a member of the tunnel, metal dependent mRNA 5' triphosphatase family which differs structurally and mechanistically from human metal independent mRNA 5' triphosphatase. In the present study the essentiality of PfPRT1 was confirmed and molecular biology tools and methods for target purification, enzymatic assessment and target engagement were developed, with the goal of running a future high throughput screening to discover PfPRT1 inhibitors., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Emerging Artemisinin (ART) resistance in Plasmodium falciparum (Pf) poses challenges for the discovery of novel drugs to tackle ART-resistant parasites. Concentrated efforts toward the ART resistance mechanism indicated a strong molecular link of ART resistance with upregulated expression of unfolded protein response pathways involving Prefoldins (PFDs). However, a complete characterization of PFDs as molecular players taking part in ART resistance mechanism, and discovery of small molecule inhibitors to block this process have not been identified to date. Here, we functionally characterized all Pf Prefoldin subunits (PFD1-6) and established a causative role played by PFDs in ART resistance by demonstrating their expression in intra-erythrocytic parasites along with their interactions with Kelch13 protein through immunoprecipitation coupled MS/MS analysis. Systematic biophysical interaction analysis between all subunits of PFDs revealed their potential to form a complex. The role of PFDs in ART resistance was confirmed in orthologous yeast PFD6 mutants, where PfPFD6 expression in yeast mutants reverted phenotype to ART resistance. We identified an FDA-approved drug "Biperiden" that restricts the formation of Prefoldin complex and inhibits its interaction with its key parasite protein substrates, MSP-1 and α-tubulin-I. Moreover, Biperiden treatment inhibits the parasite growth in ART-sensitive Pf3D7 and resistant Pf3D7k13 R539T strains. Ring survival assays that are clinically relevant to analyze ART resistance in Pf3D7k13 R539T parasites demonstrate the potency of BPD to inhibit the growth of survivor parasites. Overall, our study provides the first evidence of the role of PfPFDs in ART resistance mechanisms and opens new avenues for the management of resistant parasites., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Owing to its life cycle involving multiple hosts and species-specific biological complexities, a vaccine against Plasmodium , the causative agent of Malaria remains elusive. This makes chemotherapy the only viable means to address the clinical manifestations and spread of this deadly disease. However, rapid surge in antimalarial resistance poses significant challenges to our efforts to eliminate Malaria since the best drug available to-date; Artemisinin and its combinations are also rapidly losing efficacy. Sodium ATPase ( Pf ATP4) of Plasmodium has been recently explored as a suitable target for new antimalarials such as Cipargamin . Prior studies showed that multiple compounds from the Medicines for Malaria Venture (MMV) chemical libraries were efficient Pf ATP4 inhibitors. In this context, we undertook a structure- based virtual screening approach combined to Molecular Dynamic (MD) simulations to evaluate whether new molecules with binding affinity towards Pf ATP4 could be identified from the Pandemic Response Box (PRB), a 400-compound library of small molecules launched in 2019 by MMV. Our analysis identified new molecules from the PRB library that showed affinity for distinct binding sites including the previously known G358 site, several of which are clinically used anti-bacterial (MMV1634383, MMV1634402), antiviral (MMV010036, MMV394033) or antifungal (MMV1634494) agents. Therefore, this study highlights the possibility of exploiting PRB molecules against Malaria through abrogation of Pf ATP4 activity.Communicated by Ramaswamy H. Sarma.

Plasmodium falciparum aminoacyl tRNA synthetases (PfaaRSs) are potent antimalarial targets essential for proteome fidelity and overall parasite survival in every stage of the parasite's life cycle. So far, some of these proteins have been singly targeted yielding inhibitor compounds that have been limited by incidences of resistance which can be overcome via pan-inhibition strategies. Hence, herein, for the first time, we report the identification and in vitro antiplasmodial validation of Mitomycin (MMC) as a probable pan-inhibitor of class 1a (arginyl(A)-, cysteinyl(C), isoleucyl(I)-, leucyl(L), methionyl(M), and valyl(V)-) PfaaRSs which hypothetically may underlie its previously reported activity on the ribosomal RNA to inhibit protein translation and biosynthesis. We combined multiple in silico structure-based discovery strategies that first helped identify functional and druggable sites that were preferentially targeted by the compound in each of the plasmodial proteins: Ins1-Ins2 domain in Pf-ARS; anticodon binding domain in Pf-CRS; CP1-editing domain in Pf-IRS and Pf-MRS; C-terminal domain in Pf-LRS; and CP-core region in Pf-VRS. Molecular dynamics studies further revealed that MMC allosterically induced changes in the global structures of each protein. Likewise, prominent structural perturbations were caused by the compound across the functional domains of the proteins. More so, MMC induced systematic alterations in the binding of the catalytic nucleotide and amino acid substrates which culminated in the loss of key interactions with key active site residues and ultimate reduction in the nucleotide-binding affinities across all proteins, as deduced from the binding energy calculations. These altogether confirmed that MMC uniformly disrupted the structure of the target proteins and essential substrates. Further, MMC demonstrated IC 50  < 5 μM against the Dd2 and 3D7 strains of parasite making it a good starting point for malarial drug development. We believe that findings from our study will be important in the current search for highly effective multi-stage antimalarial drugs., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Plasmodium falciparum malaria remains a dominant infectious disease that affects Africa than the rest of the world, considering its associated cases and death rates. It's a febrile illness that produces several reliable biomarkers, for example, P. falciparum lactate dehydrogenase (PfLDH), P. falciparum Plasmodium glutamate dehydrogenase (PfGDH), and P. falciparum histidine-rich proteins (HRP-II) in blood circulatory system that can easily be employed as targets in rapid diagnostic tests (RDTs). In recent times, several DNA aptamers have been developed via SELEX technology to detect some specific malaria biomarkers (PfLDH, PvLDH, HRP-II, PfGDH) in a biosensor mode with good binding affinity properties to overcome the trend of cross-reactivity, limited sensitivity and stability problems that have been observed with immunodiagnostics. In this review, we summarized existing diagnostic methods and relevant biomarkers to suggest promising approaches to develop sensitive and species-specific multiplexed diagnostic devices enabling effective detection of malaria in complex biological matrices and surveillance in the endemic region., (© 2024 John Wiley & Sons Ltd.)

Understanding the mechanisms of drug action in malarial parasites is crucial for the development of new drugs to combat infection and to counteract drug resistance. Proteomics is a widely used approach to study host-pathogen systems and to identify drug protein targets. Plasmodione is an antiplasmodial early-lead drug exerting potent activities against young asexual and sexual blood stages in vitro with low toxicity to host cells. To elucidate its molecular mechanisms, an affinity-based protein profiling (AfBPP) approach was applied to yeast and P. falciparum proteomes. New (pro-) AfBPP probes based on the 3-benz(o)yl-6-fluoro-menadione scaffold were synthesized. With optimized conditions of both photoaffinity labeling and click reaction steps, the AfBPP protocol was then applied to a yeast proteome, yielding 11 putative drug-protein targets. Among these, we found four proteins associated with oxidoreductase activities, the hypothesized type of targets for plasmodione and its metabolites, and other proteins associated with the mitochondria. In Plasmodium parasites, the MS analysis revealed 44 potential plasmodione targets that need to be validated in further studies. Finally, the localization of a 3-benzyl-6-fluoromenadione AfBPP probe was studied in the subcellular structures of the parasite at the trophozoite stage., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

The protozoan parasite Plasmodium falciparum, responsible for the deadliest form of human malaria, employs antigenic variation via monoallelic expression as a key survival strategy. The selective activation of one out of the 60-member var gene family is key to understanding the parasite's ability to cause severe disease and evade the host immune response. var gene activation is initiated by its relocation to a specialized expression site. While the perinuclear expression site (PES) plays a crucial role in enabling the expression of a single allele, the characteristics of this PES remain largely obscure. Recent breakthroughs in genome editing tools and the discovery of regulatory noncoding RNAs have shed light on this intriguing biological feature, offering significant insights into the mechanisms of pathogen virulence., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Plasmodium falciparum (P. falciparum), which causes the most severe form of malaria, if left untreated, has 24 h window in which it can cause severe illness and even death. The aim of this study was to create the most comprehensive and informative secretory-proteome possible by combining high-accuracy and high-sensitivity protein identification technology. In this study, we used Plasmodium falciparum 3D7 (Pf3D7) as the model parasite to develop a label-free quantification proteomic strategy with the main goal of identifying Pf3D7 proteins that are supposed to be secreted outside the infected erythrocytes in the spent media culture during the in-vitro study. The spent culture media supernatant was subjected to differential and ultra-centrifugation steps followed by total protein extraction, estimation, and in-solution digestion using trypsin, digested peptides were analyzed using Nano-LC coupled with ESI for MS/MS. MS/MS spectra were processed using Maxquant software (v2.1.4.0.). Non-infected erythrocytes incubated spent cultured media supernatant were considered as control. Out of discovered 38 proteins, proteins belonging to P. falciparum spp. were EGF-like protein (C0H544), Endoplasmic reticulum chaperone GRP170 (C0H5H0), Small GTP-binding protein sar1 (Q8I1S0), Erythrocyte membrane protein 1, PfEMP1 (Q8I639), aldehyde reductase (Q8ID61), Conserved Plasmodium proteins (Q8IEH3, Q8ILD1), Antigen 332, DBL-like protein (Q8IHN4), Fe-S cluster assembly protein (Q8II78), identified and chosen for further in-depth investigation. This study highlights the value of secretory Plasmodium proteins play crucial roles in various aspects of the disease progression and host-pathogen interactions which can serve as diagnostic markers for malaria infection., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The human malaria parasite Plasmodium falciparum genome is among the most A + T rich, with low complexity regions (LCRs) inserted in coding sequences including those for proteins targeted to its essential relict plastid (apicoplast). Replication of the apicoplast genome (plDNA), mediated by the atypical multifunctional DNA polymerase PfPrex, would require additional enzymatic functions for lagging strand processing. We identified an apicoplast-targeted, [4Fe-4S]-containing, FEN/Exo (PfExo) with a long LCR insertion and detected its interaction with PfPrex. Distinct from other known exonucleases across organisms, PfExo recognized a wide substrate range; it hydrolyzed 5'-flaps, processed dsDNA as a 5'-3' exonuclease, and was a bipolar nuclease on ssDNA and RNA-DNA hybrids. Comparison with the rodent P. berghei ortholog PbExo, which lacked the insertion and [4Fe-4S], revealed interspecies functional differences. The insertion-deleted PfExoΔins behaved like PbExo with a limited substrate repertoire because of compromised DNA binding. Introduction of the PfExo insertion into PbExo led to gain of activities that the latter initially lacked. Knockout of PbExo indicated essentiality of the enzyme for survival. Our results demonstrate the presence of a novel apicoplast exonuclease with a functional LCR that diversifies substrate recognition, and identify it as the candidate flap-endonuclease and RNaseH required for plDNA replication and maintenance., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Piperaquine (PPQ) is widely used in combination with dihydroartemisinin as a first-line treatment against malaria. Multiple genetic drivers of PPQ resistance have been reported, including mutations in the Plasmodium falciparum chloroquine resistance transporter ( pfcrt ) and increased copies of plasmepsin II/III ( pm2/3 ). We generated a cross between a Cambodia-derived multidrug-resistant KEL1/PLA1 lineage isolate (KH004) and a drug-susceptible Malawian parasite (Mal31). Mal31 harbors a wild-type (3D7-like) pfcrt allele and a single copy of pm2/3 , while KH004 has a chloroquine-resistant (Dd2-like) pfcrt allele with an additional G367C substitution and multiple copies of pm2/3 . We recovered 104 unique recombinant parasites and examined a targeted set of progeny representing all possible combinations of variants at pfcrt and pm2/3 . We performed a detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes with these progenies, including PPQ survival assay, area under the dose response curve, and a limited point IC 50 . We find that inheritance of the KH004 pfcrt allele is required for reduced PPQ sensitivity, whereas copy number variation in pm2/3 further decreases susceptibility but does not confer resistance in the absence of additional mutations in pfcrt . A deep investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributions of pfcrt , pm2/3 , and parasite genetic background to a range of PPQ-related traits. Additionally, we find that the resistance phenotype associated with parasites inheriting the G367C substitution in pfcrt is consistent with previously validated PPQ resistance mutations in this transporter.IMPORTANCEResistance to piperaquine, used in combination with dihydroartemisinin, has emerged in Cambodia and threatens to spread to other malaria-endemic regions. Understanding the causal mutations of drug resistance and their impact on parasite fitness is critical for surveillance and intervention and can also reveal new avenues to limiting the evolution and spread of drug resistance. An experimental genetic cross is a powerful tool for pinpointing the genetic determinants of key drug resistance and fitness phenotypes and has the distinct advantage of quantifying the effects of naturally evolved genetic variation. Our study was strengthened since the full range of copies of KH004 pm2/3 was inherited among the progeny clones, allowing us to directly test the role of the pm2/3 copy number on resistance-related phenotypes in the context of a unique pfcrt allele. Our multigene model suggests an important role for both loci in the evolution of this multidrug-resistant parasite lineage., Competing Interests: The authors declare no conflict of interest.

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., (© 2024. The Author(s).)

Plasmodium falciparum causes severe malaria and assembles a protein translocon (PTEX) complex at the parasitophorous vacuole membrane (PVM) of infected erythrocytes, through which several hundred proteins are exported to facilitate growth. The preceding liver stage of infection involves growth in a hepatocyte-derived PVM; however, the importance of protein export during P. falciparum liver infection remains unexplored. Here, we use the FlpL/ FRT system to conditionally excise genes in P. falciparum sporozoites for functional liver-stage studies. Disruption of PTEX members ptex150 and exp2 did not affect sporozoite development in mosquitoes or infectivity for hepatocytes but attenuated liver-stage growth in humanized mice. While PTEX150 deficiency reduced fitness on day 6 postinfection by 40%, EXP2 deficiency caused 100% loss of liver parasites, demonstrating that PTEX components are required for growth in hepatocytes to differing degrees. To characterize PTEX loss-of-function mutations, we localized four liver-stage Plasmodium export element (PEXEL) proteins. P. falciparum liver specific protein 2 (LISP2), liver-stage antigen 3 (LSA3), circumsporozoite protein (CSP), and a Plasmodium berghei LISP2 reporter all localized to the periphery of P. falciparum liver stages but were not exported beyond the PVM. Expression of LISP2 and CSP but not LSA3 was reduced in ptex150-FRT and exp2-FRT liver stages, suggesting that expression of some PEXEL proteins is affected directly or indirectly by PTEX disruption. These results show that PTEX150 and EXP2 are important for P. falciparum development in hepatocytes and emphasize the emerging complexity of PEXEL protein trafficking., Competing Interests: Competing interests statement:The authors declare no competing interest.