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We previously reported that the Plasmodium falciparum putative serine/threonine protein phosphatase 7 (PP7) is a high-confidence substrate of the cAMP-dependent protein kinase (PKA). Here we explore the function of PP7 in asexual P. falciparum blood stage parasites. We show that conditional disruption of PP7 leads to a severe growth arrest. We show that PP7 is a calcium-dependent phosphatase that interacts with calmodulin and calcium-dependent protein kinase 1 (CDPK1), consistent with a role in calcium signaling. Notably, PP7 was found to be dispensable for erythrocyte invasion, but was crucial for ring-stage development, with PP7-null parasites arresting shortly following invasion and showing no transition to ameboid forms. Phosphoproteomic analysis revealed that PP7 may regulate certain PKAc substrates. Its interaction with calmodulin and CDPK1 further emphasizes a role in calcium signaling, while its impact on early ring development and PKAc substrate phosphorylation underscores its importance in parasite development., Importance: Plasmodium falciparum causes malaria and is responsible for more than 600,000 deaths each year. Although effective drugs are available to treat disease, the spread of drug-resistant parasites endangers their future efficacy. It is hoped that a better understanding of the biology of malaria parasites will help us to discover new drugs to tackle the resistance problem. Our work is focused on the cell signaling mechanisms that control the development of the parasite throughout its complex life cycle. All signal transduction pathways are ultimately regulated by reversible protein phosphorylation by protein kinase and protein phosphatase enzymes. In this study, we investigate the function of calcium-dependent protein phosphatase PP7 and show that it is essential for the development of ring-stage parasites following the invasion of human erythrocytes. Our results contribute to the understanding of the erythrocytic stages of the parasite life cycle that cause malaria pathology., Competing Interests: The authors declare no conflict of interest.

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.

Targeted nanodrug delivery systems are highly anticipated for the treatment of malaria. It is known that Plasmodium can induce new permeability pathways (NPPs) on the membrane of infected red blood cells (iRBCs) for their nutrient uptake. The NPPs also enable the uptake of nanoparticles (NPs) smaller than 80 nm. Additionally, Plasmodium maintains a stable, slightly acidic, and reductive internal environment with higher glutathione (GSH) levels. Based on this knowledge, methyl artelinate (MA, a prodrug-like derivative of dihydroartemisinin) nanoparticles (MA-PCL-NPs) were developed using poly(ethylene glycol)- b -poly(ε-caprolactone) (mPEG-PCL) by a thin-film dispersion method and were further coated with polydopamine (PDA) to obtain MA-PCL@PDA-NPs with a particle size of ∼30 nm. The biomaterial PDA can be degraded in slightly acidic and reductive environments, thereby serving as triggers for drug release. MA could generate reactive oxygen species and decrease GSH levels, consequently causing parasite damage. The in vitro release experiment results indicated that the cumulative release percentage of MA from MA-PCL@PDA-NPs was considerably higher in phosphate buffer with 10 mM GSH at pH 5.5 (88.10%) than in phosphate buffer without GSH at pH 7.4 (16.98%). The green fluorescence within iRBCs of coumarin 6, the probe of NPs (C6-PCL@PDA-NPs), could be reduced significantly after adding the NPP inhibitor furosemide ( p < 0.001), which demonstrated that MA-PCL@PDA-NPs could be ingested into iRBCs through NPPs. In vivo antimalarial pharmacodynamics in Plasmodium berghei K173-bearing mice showed that the inhibition ratio of MA-PCL@PDA-NPs (93.96%) was significantly higher than that of commercial artesunate injection (AS-Inj, 63.33%). The above results showed that the developed MA-PCL@PDA-NPs possessed pH-GSH dual-responsive drug release characteristics and targeting efficacy for iRBCs, leading to higher antimalarial efficacy against Plasmodium .

Leishmaniasis is a neglected tropical disease caused by parasites of the genus Leishmania and is responsible for more than 1 million new cases and 70,000 deaths annually worldwide. Treatment has high costs, toxicity, complex and long administration time, several adverse effects, and drug-resistant strains, therefore new therapies are urgently needed. Synthetic compounds have been highlighted in the medicinal chemistry field as a strong option for drug development against different diseases. Organic salts (OS) have multiple biological activities, including activity against protozoa such as Leishmania spp. This study aimed to investigate the in vitro leishmanicidal activity and death mechanisms of a thiohydantoin salt derived from l-arginine (ThS) against Leishmania amazonensis. We observed that ThS treatment inhibited promastigote proliferation, increased ROS production, phosphatidylserine exposure and plasma membrane permeabilization, loss of mitochondrial membrane potential, lipid body accumulation, autophagic vacuole formation, cell cycle alteration, and morphological and ultrastructural changes, showing parasites death. Additionally, ThS presents low cytotoxicity in murine macrophages (J774A.1), human monocytes (THP-1), and sheep erythrocytes. ThS in vitro cell treatment reduced the percentage of infected macrophages and the number of amastigotes per macrophage by increasing ROS production and reducing TNF-α levels. These results highlight the potential of ThS among thiohydantoins, mainly related to the arginine portion, as a leishmanicidal drug for future drug strategies for leishmaniasis treatment. Notably, in silico investigation of key targets from L. amazonensis, revealed that a ThS compound from the l-arginine amino acid strongly interacts with arginase (ARG) and TNF-α converting enzyme (TACE), suggesting its potential as a Leishmania inhibitor., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: 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. If there are other authors, they 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 Elsevier B.V. All rights reserved.)

Babesia spp. are obligate intracellular parasites that invade host cells to complete their asexual development and transmission. Here, we identified a transcription factor AP2-M (BXIN&#95;0799) in Babesia sp. Xinjiang (Bxj), a member of the Apicomplexan AP2 family, which regulates gene expression related to red blood cell (RBC) invasion and cell cycle progression. Our genome-wide analysis of (Cut-Tag) data shows that AP2-M specifically recognized DNA motifs in the promoters of target genes. AP2-M target genes included other AP2 gene family members and epigenetic markers, which could modulate gene expression involved in RBC invasion, merozoite morphology, and cell cycle phases, as indicated by RNA sequencing, proteomics, and single-cell RNA sequencing (scRNA-seq) data from an ap2-m gene disrupted strain (AP2-M (-)). We conclude that AP2-M appeared to contribute to the process of red blood cell invasion, maintain merozoite morphology, and cell cycle progression through GS and MS phases., (© 2024 Federation of American Societies for Experimental Biology.)

Current malaria diagnostics are invasive, lack sensitivity, and rapid tests are plagued by deletions in target antigens. Here we introduce the Cytophone, an innovative photoacoustic flow cytometer platform with high-pulse-rate lasers and a focused ultrasound transducer array to noninvasively detect and identify malaria-infected red blood cells (iRBCs) using specific wave shapes, widths, and time delays generated from the absorbance of laser energy by hemozoin, a universal biomarker of malaria infection. In a population of Cameroonian adults with uncomplicated malaria, we assess our device for safety in a cross-sectional cohort (n = 10) and conduct a performance assessment in a longitudinal cohort (n = 20) followed for 30 ± 7 days after clearance of parasitemia. Longitudinal cytophone measurements are compared to point-of-care and molecular assays (n = 94). Cytophone is safe with 90% sensitivity, 69% specificity, and a receiver-operator-curve-area-under-the-curve (ROC-AUC) of 0.84, as compared to microscopy. ROC-AUCs of Cytophone, microscopy, and RDT compared to quantitative PCR are not statistically different from one another. The ability to noninvasively detect iRBCs in the bloodstream is a major advancement which offers the potential to rapidly identify both the large asymptomatic reservoir of infection, as well as diagnose symptomatic cases without the need for a blood sample., (© 2024. The Author(s).)

Lipid droplets (LDs) are organelles that are central to lipid and energy homeostasis across all eukaryotes. In the malaria-causing parasite Plasmodium falciparum the roles of LDs in lipid acquisition from its host cells and their metabolism are poorly understood, despite the high demand for lipids in parasite membrane synthesis. We systematically characterised LD size, composition and dynamics across the disease-causing blood infection. Applying split fluorescence emission analysis and three-dimensional (3D) focused ion beam-scanning electron microscopy (FIB-SEM), we observed a decrease in LD size in late schizont stages. LD contraction likely signifies a switch from lipid accumulation to lipid utilisation in preparation for parasite egress from host red blood cells. We demonstrate connections between LDs and several parasite organelles, pointing to potential functional interactions. Chemical inhibition of triacylglyerol (TAG) synthesis or breakdown revealed essential LD functions for schizogony and in counteracting lipid toxicity. The dynamics of lipid synthesis, storage and utilisation in P. falciparum LDs might provide a target for new anti-malarial intervention strategies., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Treatment failure with amodiaquine was reported in Dangassa, where red blood cell (RBC) polymorphisms are found and seasonal malaria chemoprevention (SMC) is underway. Here, we aimed at assessing the influence of RBC polymorphisms on SMC effectiveness. This was a secondary analysis of data from a study conducted in Dangassa. Children aged 5 to 14 years enrolled in an open randomized study were assigned either to receive SMC (intervention arm) or not (control arm). SMC was implemented from July to November. For all children, hemoglobin type and blood group were determined at enrolment in July, and parasitemia and hemoglobin level were monthly monitored by finger-prick. Overall, 166 children were enrolled among which 82 (49.40%) in the control arm and 84 (50.60%) in the SMC arm. The prevalence of HbAS was 10.24% (17/166) with 12.20% in the control and 8.33% in the SMC arm. O group was the most common overall (45%) and in the SMC arm (54%), but the control arm had more B (39.02%) than O (36.59%). In the SMC arm, no case of Plasmodium infection and malaria disease was observed in the 7 HbAS children while in Non-HbAS children, peaks of infection and disease prevalence were respectively observed in October (24.66%) and November (7.14%). For the SMC arm, in group O and Non-group O, Plasmodium infection cases were observed from August to December. Plasmodium infection and malaria disease were more frequently observed in HbAS children in the control arm than in the SMC arm. Further studies are needed to assess factors associated with the asymptomatic carriage of parasites during SMC in Dangassa. NCT04149106., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.)

Background: This study examines the feasibility and effects of introducing microRNA mimic into red blood cells (RBCs) at the initial phases of Plasmodium falciparum 3D7 (Pf3D7) infection. The aim is to determine the correlation between increased expression of miR-451a and parasitaemia., Methods: In this study miR-mimic-451a labelled with Cy3 and transfected into control and infected RBCs using lipofectamine and analysed using the fluorescence microscopy and flow cytometry. The study demonstrated the efficacy of miR-451a by treating pre-and post-transfected control RBCs and Pf3D7-infected RBCs with miR-mimic-451a. We also examined its impact on % growth inhibition of Pf3D7, oxidative stress markers (Luminometry, LPO, SOD, CAT, GSH and GPx). Additionally, determination of pH, haemoglobin (Hb), and proteomic profile performed using SDS-PAGE., Results: Modified expression level of mir-451a has the potential to change the progression of the infection and yielded a 50% decrease in parasitaemia within 48 h. Moreover, transfected samples were shown to be efficacious in counteracting the oxidative stress-induced alterations during Pf3D7 infection and enable to return the cells towards the normalcy. Modified proteomic profile of transfected iRBCs demonstrates the correlation between overexpression of miRNA and protein expression. where, the major changes were observed in the heavy molecular weight proteins more than 57 kDa., Conclusion: The study reveals promising effects of miR-mimic-451a enrichment during RBC stages of Pf3D7, offering insights into potential malaria therapeutic strategies and potential biomedical research implications., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Equine piroplasmosis (EP) is a protozoal disease affecting equids, caused by Theileria equi and Babesia caballi . EP is conventionally diagnosed using microscopic, molecular, and/or serological methods, which are time-consuming. Consequently, there is a need for faster testing methods. In this study, we evaluated the application of the Sysmex XN-31 automated hematology analyzer, originally a rapid test for detecting malaria in humans, for the diagnosis of EP. The cultured parasites were measured using the XN-31 that had been customized for horse blood samples (XN-31m). The following parameters were evaluated: limit of detection (LoD), limit of quantification (LoQ), linearity, carryover, precision, and correlation with microscopic examination. The XN-31m detected infected red blood cells (RBCs) in approximately 1 minute. The LoD and LoQ for B. caballi were 4.54 infected RBCs/μL and 14.10 infected RBCs/μL, while those for T. equi were 5.80 infected RBCs/μL and 11.44 infected RBCs/μL, respectively. Linearity showed excellent correlation ( R 2 > 0.99), and carryover never exceeded 0.5%. The coefficient of variation was under 5%. The correlation between the results obtained using XN-31m and microscopic examination was high ( R 2 > 0.98). In conclusion, the XN-31 analyzer detected B. caballi and T. equi parasites in approximately 1 minute with high sensitivity. The results indicate the potential of the XN-31 analyzer as a fast and user-friendly diagnostic method for EP., Importance: In this study, we demonstrated that the automated hematology analyzer, XN-31, can detect red blood cells infected with Babesia caballi and Theileria equi in about 1 minute. We evaluated the diagnostic performance of the XN-31 analyzer for equine piroplasmosis, providing evidence of its potential as a diagnostic tool for this disease., Competing Interests: Yuji Toya, Mikako Sengoku, and Seiichiro Tsuchiya are employees of Sysmex corp.

Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) is a unique asexual blood-stage malaria vaccine candidate because of its high conservation and essential biological function of binding to basigin on the erythrocyte surface. Recent studies by Barrett et al., Wang et al., and King et al., have brought RH5-based vaccine development a step forward based on a rational antigen design strategy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background and Objectives: Plasmodium species are naturally transmitted by Anopheles mosquitos. The parasite infects red blood cells (RBCs) and can be transfused with blood products. In non-endemic areas, the main risk of infection arises from travellers coming back and people immigrating from malaria-endemic regions. Endemic countries face a permanent risk of infection from transfusion-transmitted malaria (TTM). TTM may cause life-threatening complications in patients dependent on blood donations. This study aimed to investigate the efficacy of Plasmodium falciparum inactivation in RBC units by treatment with short-wavelength ultraviolet C (UVC) light in the absence of photochemical additives., Materials and Methods: RBC units were spiked with P. falciparum to a parasite density of 0.1%-1% and irradiated with up to 4.5 J/cm 2 UVC. The parasite density of UVC-treated dilution series and untreated controls were compared over 3 weeks after irradiation., Results: The lowest dose of 1.5 J/cm 2 UVC led to a 3.1 log reduction in parasite load compared with the untreated control. The inactivation capacity was dose-dependent. Strikingly, 4.5 J/cm 2 led to ≥5.3 log unit reduction, which was equivalent to a complete inactivation in two out of three experiments., Conclusion: Pathogen reduction with UVC light was previously shown to be effective for different bacteria and viruses, but the inactivation of parasites in RBC concentrates was not addressed until now. The present study provides evidence for significant inactivation of P. falciparum-infected RBCs by UVC light., (© 2024 The Author(s). Vox Sanguinis published by John Wiley & Sons Ltd on behalf of International Society of Blood Transfusion.)

In vitro modification of Plasmodium falciparum genes is the cornerstone of basic and translational malaria research. Achieved through DNA transfection, these modifications may entail altering protein sequence or abundance. Such experiments are critical for defining the molecular mechanisms of key parasite phenotypes and for validation of drug and vaccine targets. Despite its importance, successful transfection remains difficult and is a resource-intensive, rate-limiting step in P. falciparum research. Here, we report that inefficient loading of plasmid into erythrocytes limits transfection efficacy with commonly used electroporation methods. As these methods also require expensive instrumentation and consumables that are not broadly available, we explored a simpler method based on plasmid loading through hypotonic lysis and resealing of erythrocytes. We used parasite expression of a sensitive NanoLuc reporter for rapid evaluation and optimization of each step. Hypotonic buffer composition, resealing buffer volume and composition, and subsequent incubation affected plasmid retention and successful transfection. While ATP was critical for erythrocyte resealing, addition of Ca ++ or glutathione did not improve transfection efficiency, with increasing Ca ++ concentrations proving detrimental to outcomes. Compared with either the standard electroporation method or a previously reported hypotonic loading protocol, the optimized method yields greater plasmid loading and higher expression of the NanoLuc reporter 48 h after transfection. It also produced significantly faster outgrowth of parasites in transfections utilizing either episomal expression or CRISPR-Cas9 mediated integration. This new method produces higher P. falciparum transfection efficiency, reduces resource requirements and should accelerate molecular studies of malaria drug and vaccine targets., 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., (Published by Elsevier Ltd.)

Objectives: Human babesiosis is an emerging and potentially fatal tick-borne disease caused by intraerythrocytic parasites of the Babesia genus. Among these, Babesia duncani is particularly notable for causing severe and life-threatening illness in humans. Accurate diagnosis and effective disease management hinge on the detection of active B. duncani infections. While molecular assays are available to detect the parasite in blood, a reliable method for identifying biomarkers of active infection remains elusive., Methods: We developed the first B. duncani antigen capture assays, targeting two immunodominant antigens, BdV234 and BdV38. These assays were validated using established in vitro and in vivo B. duncani infection models, and following drug treatment., Results: The assays demonstrated no cross-reactivity with other species such as B. microti, B. divergens, Babesia MO1, or Plasmodium falciparum, and can detect as few as 115 infected erythrocytes/µl of blood. Screening of 1731 blood samples from various biorepositories, including samples previously identified as Lyme and/or B. microti-positive, as well as new specimens from wild mice, revealed no evidence of B. duncani infection or cross-reactivity., Conclusions: These assays hold significant promise for various applications, including point-of-care testing for the early detection of B. duncani in patients, field tests for screening reservoir hosts, and high-throughput screening of blood samples intended for transfusion., Competing Interests: Declarations of competing interest The authors have no competing interests to declare., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Malaria is a deadly disease of significant concern for the international community. It is an infectious disease caused by a Plasmodium spp. parasite and transmitted by the bite of an infected female Anopheles mosquito. The parasite multiplies in the liver and then destroys the person's red blood cells until it reaches the severe stage, leading to death. The most used tools for diagnosing this disease are the microscope and the rapid diagnostic test (RDT), which have limitations preventing control of the disease. Computer vision technologies present alternatives by providing the means for early detection of this disease before it reaches the severe stage, facilitating treatment and saving patients. In this article, we suggest deep learning methods for earlier and more accurate detection of malaria parasites with high generalization capabilities using microscopic images of blood smears from many heterogeneous patients. These techniques are based on an image preprocessing method that mitigates some of the challenges associated with the variety of red cell characteristics due to patient diversity and other artifacts present in the data. For the study, we collected 65,970 microscopic images from 876 different patients to form a dataset of 33,007 images with a variety that enables us to create models with a high level of generalization. Three types of convolutional neural networks were used, namely Convolutional Neural Network (CNN), DenseNet, and LeNet-5, and the highest classification accuracy on the test data was 97.50% found with the DenseNet model., 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 Elsevier B.V. All rights reserved.)

In humans and Drosophila melanogaster , the functional convergence of the endosomal sorting complex required for transport (ESCRT) machinery that is in charge of selecting ubiquitinated proteins for sorting into multivesicular bodies, and the retromer, that is the complex responsible for protein recycling to the plasma membrane and Golgi apparatus. ESCRT and retromer complexes are codependent for protein sorting recycling, degradation, and secretion. In this article, we studied the EhVps35 C isoform (referred to as EhVps35), that is the central member of the Entamoeba histolytica retromer, and its relation with the ESCRT machinery during sorting and protein recycling events and their involvement virulence. Our findings revealed that EhVps35 interacts with at least 300 proteins that participate in multiple cellular processes. Laser confocal and transmission electronic microscopy images, as well as secretion assays, revealed that EhVps35 is secreted in vesicles together with EhVps23 and EhADH (both ESCRT machinery proteins). In addition, immunoprecipitation, immunofluorescence, and molecular docking assays revealed the relationship among EhVps35 and other ESCRT machinery proteins. Red blood cell stimulus increased EhVps35 secretion, and the knockdown of the Ehvps35 gene in trophozoites reduced their capacity to migrate and invade tissues. This also impacts the cellular localization of ubiquitin, EhVps23 (ESCRT-I), and EhVps32 (ESCRT-III) proteins, strongly suggesting their functional relationship. Our results, taken together, give evidence that EhVps35 is a key factor in E. histolytica virulence mechanisms and that it, together with the ESCRT machinery components and other regulatory proteins, is involved in vesicle trafficking, secretion, migration, and cell proliferation., Competing Interests: The 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 Díaz-Valdez, Javier-Reyna, Galindo, Salazar-Villatoro, Montaño and Orozco.)

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.)

Plasmodium falciparum undergoes sequestration within deep tissues of the human body, spanning multiple organ systems with differing oxygen (O2) concentrations. The parasite is exposed to an even greater range of O2 concentrations as it transitions from the human to the mosquito host, suggesting a high level of plasticity as it navigates these different environments. In this review, we explore factors that may contribute to the parasite's response to different environmental O2 concentrations, recognizing that there are likely multiple pieces to this puzzle. We first review O2-sensing mechanisms, which exist in other apicomplexans such as Toxoplasma gondii and consider whether similar systems could exist in Plasmodium. Next, we review morphological and functional changes in P. falciparum's mitochondrion during the asexual-to-sexual stage transition and discuss how these changes overlap with the parasite's access to O2. We then delve into reactive oxygen species (ROS) as ROS production is influenced by O2 availability and oxidative stress impacts Plasmodium intraerythrocytic development. Lastly, given that the primary role of the red blood cell (RBC) is to deliver O2 throughout the body, we discuss how changes in the oxygenation status of hemoglobin, the RBC's O2-carrying protein and key nutrient for Plasmodium, could also potentially impact the parasite's growth during intraerythrocytic development. This review also highlights studies that have investigated P. falciparum biology under varying O2 concentrations and covers technical aspects related to P. falciparum cultivation in the lab, focusing on sources of technical variation that could alter the amount of dissolved O2 encountered by cells during in vitro experiments. Lastly, we discuss how culture systems can better replicate in vivo heterogeneity with respect to O2 gradients, propose ideas for further research in this area, and consider translational implications related to O2 and malaria., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Nahid 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.)

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.)

Background: Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins are expressed on the surface of infected erythrocytes, mediating parasite sequestration in the vasculature. PfEMP1 is a major target of protective antibodies, but the features of the antibody response are poorly defined., Methods: In Malawian children with cerebral or uncomplicated malaria, we characterized the antibody response to 39 recombinant PfEMP1 Duffy binding like (DBL) domains or cysteine-rich interdomain regions (CIDRs) in detail, including measures of antibody classes, subclasses, and engagement with Fcγ receptors and complement. Using elastic net regularized logistic regression, we identified a combination of seven antibody targets and Fc features that best distinguished between children with cerebral and uncomplicated malaria. To confirm the role of the selected targets and Fc features, we measured antibody-dependent neutrophil and THP-1 cell phagocytosis of intercellular adhesion molecule-1 (ICAM-1) and endothelial protein C (EPCR) co-binding infected erythrocytes., Results: The selected features distinguished between children with cerebral and uncomplicated malaria with 87% accuracy (median, 80-96% interquartile range) and included antibody to well-characterized DBLβ3 domains and a less well-characterized CIDRγ12 domain. The abilities of antibodies to engage C1q and FcγRIIIb, rather than levels of IgG, correlated with protection. In line with a role of FcγRIIIb binding antibodies to DBLβ3 domains, antibody-dependent neutrophil phagocytosis of ICAM-1 and EPCR co-binding IE was higher in uncomplicated malaria (15% median, 8-38% interquartile range) compared to cerebral malaria (7%, 30-15%, p < 0.001)., Conclusions: Antibodies associated with protection from cerebral malaria target a subset of PfEMP1 domains. The Fc features of protective antibody response include engagement of FcγRIIIb and C1q, and ability to induce antibody-dependent neutrophil phagocytosis of infected erythrocytes. Identifying the targets and Fc features of protective immunity could facilitate the development of PfEMP1-based therapeutics for cerebral malaria., (© 2024. The Author(s).)

Rodent malaria models serve as important preclinical antimalarial and vaccine testing tools. Evaluating treatment outcomes in these models often requires manually counting parasite-infected red blood cells (iRBCs), a time-consuming process, which can be inconsistent between individuals and laboratories. We have developed an easy-to-use machine learning (ML)-based software, Malaria Screener R, to expedite and standardize such studies by automating the counting of Plasmodium iRBCs in rodents. This software can process Giemsa-stained blood smear images captured by any camera-equipped microscope. It features an intuitive graphical user interface that facilitates image processing and visualization of the results. The software has been developed as a desktop application that processes images on standard Windows and MacOS computers. A previous ML model created by the authors designed to count Plasmodium falciparum-infected human RBCs did not perform well counting Plasmodium-infected mouse RBCs. We leveraged that model by loading the pretrained weights and training the algorithm with newly collected data to target Plasmodium yoelii- and Plasmodium berghei-infected mouse RBCs. This new model reliably measured both P. yoelii and P. berghei parasitemia (R2 = 0.9916). Additional rounds of training data to incorporate variances due to length of Giemsa staining and type of microscopes, etc., have produced a generalizable model, meeting WHO competency level 1 for the subcategory of parasite counting using independent microscopes. Reliable, automated analyses of blood-stage parasitemia will facilitate rapid and consistent evaluation of novel vaccines and antimalarials across laboratories in an easily accessible in vivo malaria model.

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.)

The highly conserved and essential Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has emerged as the leading target for vaccines against the disease-causing blood stage of malaria. However, the features of the human vaccine-induced antibody response that confer highly potent inhibition of malaria parasite invasion into red blood cells are not well defined. Here, we characterize 236 human IgG monoclonal antibodies, derived from 15 donors, induced by the most advanced PfRH5 vaccine. We define the antigenic landscape of this molecule and establish that epitope specificity, antibody association rate, and intra-PfRH5 antibody interactions are key determinants of functional anti-parasitic potency. In addition, we identify a germline IgG gene combination that results in an exceptionally potent class of antibody and demonstrate its prophylactic potential to protect against P. falciparum parasite challenge in vivo. This comprehensive dataset provides a framework to guide rational design of next-generation vaccines and prophylactic antibodies to protect against blood-stage malaria., Competing Interests: Declaration of interests J.R.B., A.J.R.C., G.G., B.G.W., L.D.W.K., L.T.W., J.T., K. McHugh, and S.J.D. are inventors on patent applications relating to RH5 malaria vaccines and/or antibodies. A.M.M. and S.J.D. have consulted to GSK on malaria vaccines. A.M.M. has an immediate family member who is an inventor on patent applications relating to RH5 malaria vaccines and antibodies., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Babesia and Plasmodium pathogens, the causative agents of babesiosis and malaria, are vector-borne intraerythrocytic protozoan parasites, posing significant threats to both human and animal health. The widespread resistance exhibited by these pathogens to various classes of antiparasitic drugs underscores the need for the development of novel and more effective therapeutic strategies. Antifolates have long been recognized as attractive antiparasitic drugs as they target the folate pathway, which is essential for the biosynthesis of purines and pyrimidines, and thus is vital for the survival and proliferation of protozoan parasites. More efficacious and safer analogs within this class are needed to overcome challenges due to resistance to commonly used antifolates, such as pyrimethamine, and to address liabilities associated with the dihydrotriazines, WR99210 and JPC-2067. Here, we utilized an in vitro culture condition suitable for the continuous propagation of Babesia duncani, Babesia divergens, Babesia MO1, and Plasmodium falciparum in human erythrocytes to screen a library of 50 dihydrotriazines and 29 biguanides for their efficacy in vitro and compared their potency and therapeutic indices across different species and isolates. We identified nine analogs that inhibit the growth of all species, including the P. falciparum pyrimethamine-resistant strain HB3, with IC 50 values below 10 nM, and display excellent in vitro therapeutic indices. These compounds hold substantial promise as lead antifolates for further development as broad-spectrum antiparasitic drugs., Competing Interests: The authors declare no conflict of interest.

Bovine lactoferrin (bLF) is a 77 kDa glycoprotein that is abundant in bovine breast milk and exerts various bioactive functions, including antibacterial and antiviral functions. Few studies have explored bLF activity against parasites. We found that bLF affects hemozoin synthesis by binding to heme, inhibiting heme iron polymerization necessary for Plasmodium berghei ANKA survival in infected erythrocytes, and also binds to hemozoin, causing it to disassemble. In a challenge test, bLF administration inhibited the growth of murine malaria parasites compared to untreated group growth. To determine whether the iron content of bLF affects the inhibition of malaria growth, we tested bLFs containing different amounts of iron (apo-bLF, native-bLF, and holo-bLF), but found no significant difference in their effects. This indicated that the active sites were located within the bLFs themselves. Further studies showed that the C-lobe domain of bLF can inhibit hemozoin formation and the growth of P. berghei ANKA. Evaluation of pepsin degradation products of the C-lobe identified a 47-amino-acid section, C-1, as the smallest effective region that could inhibit hemozoin formation. This study highlights bLF's potential as a novel therapeutic agent against malaria, underscoring the importance of its non-iron-dependent bioactive sites in combating parasite growth., (© 2024. The Author(s).)

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: 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.)

Raman spectroscopy was used to study erythrocytes collected from patients diagnosed with malaria at the University Hospital in Kraków and from healthy volunteers. A laser line with a wavelength of 442 nm was used to induce the Raman resonance of haem, while a laser with a wavelength of 785 nm was used for the normal Raman effect. The results were analysed using Principal Component Analysis. For the 442 nm laser line, analysis of the entire spectral range (3200 cm -1 to 300 cm -1 ) showed satisfactory separation of Raman spectra for healthy cells from infected cells, which was significantly improved in the 1500 cm -1 -1200 cm -1 spectral range. For the 785 nm laser line, some separation was observed in each range studied, but the best results were achieved over the full spectral range. Plasmodium-derived nucleic acids and phosphodiester vibrations were observed at excitation lines of 442 nm and 785 nm, respectively., 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 Elsevier B.V. All rights reserved.)

Invasion of human red blood cells by the malaria parasite Plasmodium falciparum is followed by dramatic modifications of erythrocytes properties, including de novo formation of new membrane systems. Lipid transfer proteins from both the parasite and the host cell are most likely an important part of those membrane remodeling processes. Using bioinformatics and in silico structural analysis, we have identified five P. falciparum potential lipid transfer proteins containing cellular retinaldehyde binding - triple functional domain (CRAL-TRIO). Two of these proteins, C6KTD4, encoded by the PF3D7&#95;0629900 gene and Q8II87, encoded by the PF3D7&#95;1127600 gene, were studied in more detail. In vitro lipid transfer assays using recombinant C6KTD4 and Q8II87 confirmed that these proteins are indeed bona fide lipid transfer proteins. C6KTD4 transfers sterols, phosphatidylinositol 4,5 bisphosphate, and, to some degree, also phosphatidylcholine between two membrane compartments. Q8II87 possesses phosphatidylserine transfer activity in vitro. In the yeast model, the expression of P. falciparumQ8II87 protein partially complements the absence of Sec14p and its closest homologue, Sfh1p. C6KTD4 protein can substitute for the collective essential function of oxysterol-binding related proteins. According to published whole genome studies in P. falciparum, absence of C6KTD4 and Q8II87 proteins has severe consequences for parasite viability. Therefore, CRAL-TRIO lipid transfer proteins of P. falciparum are potential targets of novel antimalarials, in search for which the yeast model expressing these proteins could be a valuable tool., 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 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.)

Striking morphological transformations characterize the invasion of a red blood cell by the malaria parasite. Shortly after the infection, parasite-induced membranes appear in the cytosol of the affected host erythrocyte. One intensely investigated membrane type, commonly called Maurer's clefts, has a slit-like morphology and can be arranged in the form of extended three-dimensional membrane stacks or networks. Here we report the three-dimensional reconstruction of a second membrane type, giant or extended membrane rings/loops, that have only occasionally been described on single ultrathin sections, however that have never been systematically examined so far. Serial ultrathin sectioning of P. falciparum-infected red blood cells, subsequent three-dimensional reconstructions, and in addition examination of Giemsa-stained blood films revealed that intraerythrocytic membrane rings/loops are not isolated structures but are locally in contact with the parasite. They consist either of the parasitophorous vacuolar membrane alone or contain the parasitophorous vacuolar membrane including the plasma membrane of the parasite and small amounts of parasite cytoplasm. We demonstrate that membrane rings/loops represent surface extensions of the parasite that maybe involved in ring stage parasite formation and Maurer's cleft generation at least in a subset of infected red blood cells., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Apical membrane antigen-1 (AMA1) is a conserved malarial vaccine candidate essential for the formation of tight junctions with the rhoptry neck protein (RON) complex, enabling Plasmodium parasites to invade human erythrocytes, hepatocytes, and mosquito salivary glands. Despite its critical role, extensive surface polymorphisms in AMA1 have led to strain-specific protection, limiting the success of AMA1-based interventions beyond initial clinical trials. Here, we identify an i-body, a humanised single-domain antibody-like molecule that recognises a conserved pan-species conformational epitope in AMA1 with low nanomolar affinity and inhibits the binding of the RON2 ligand to AMA1. Structural characterisation indicates that the WD34 i-body epitope spans the centre of the conserved hydrophobic cleft in AMA1, where interacting residues are highly conserved among all Plasmodium species. Furthermore, we show that WD34 inhibits merozoite invasion of erythrocytes by multiple Plasmodium species and hepatocyte invasion by P. falciparum sporozoites. Despite a short half-life in mouse serum, we demonstrate that WD34 transiently suppressed P. berghei infections in female BALB/c mice. Our work describes the first pan-species AMA1 biologic with inhibitory activity against multiple life-cycle stages of Plasmodium. With improved pharmacokinetic characteristics, WD34 could be a potential immunotherapy against multiple species of Plasmodium., (© 2024. The Author(s).)

Merozoites utilize sialic acids on the red blood cell (RBC) cell surface to rapidly adhere to and invade the RBCs. Newcastle disease virus (NDV) displays a strong affinity toward membrane-bound sialic acids. Incubation of NDV with the malaria parasites dose-dependently reduces its cellular viability. The antiplasmodial activity of NDV is specific, as incubation with Japanese encephalitis virus, duck enteritis virus, infectious bronchitis virus, and influenza virus did not affect the parasite propagation. Interestingly, NDV is reducing more than 80% invasion when RBCs are pretreated with the virus. Removal of the RBC surface proteins or the NDV coat proteins results in disruption of the virus binding to RBC. It suggests the involvement of specific protein: ligand interaction in virus binding. We established that the virus engages with the parasitized RBCs (PRBCs) through its hemagglutinin neuraminidase (HN) protein by recognizing sialic acid-containing glycoproteins on the cell surface. Blocking of the HN protein with free sialic acid or anti-HN antibodies abolished the virus binding as well as its ability to reduce parasite growth. Interestingly, the purified HN from the virus alone could inhibit the parasite's growth in a dose-dependent manner. NDV binds strongly to knobless murine parasite strain Plasmodium yoelii and restricted the parasite growth in mice. Furthermore, the virus was found to preferentially target the PRBCs compared to normal erythrocytes. Immunolocalization studies reveal that NDV is localized on the plasma membrane as well as weakly inside the PRBC. NDV causes neither any infection nor aggregation of the human RBCs. Our findings suggest that NDV is a potential candidate for developing targeted drug delivery platforms for the Plasmodium-infected RBCs., (© 2024 Federation of American Societies for Experimental Biology.)

The retromer is a cellular structure that recruits and recycles proteins inside the cell. In mammalian and yeast, the retromer components have been widely studied, but very little in parasites. In yeast, it is formed by a SNX-BAR membrane remodeling heterodimer and the cargo selecting complex (CSC), composed by three proteins. One of them, the Vps26 protein, possesses a flexible and intrinsically disordered region (IDR), that facilitates interactions with other proteins and contributes to the retromer binding to the endosomal membrane. In Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, the retromer actively participates during the high mobility and phagocytosis of trophozoites, but the molecular details in these events, are almost unknown. Here, we studied the EhVps26 role in phagocytosis. Bioinformatic analyses of EhVps26 revealed a typical arrestin folding structure of the protein, and a long and charged IDR, as described in other systems. EhVps26 molecular dynamics simulations (MDS) allowed us to predict binding pockets for EhVps35, EhSNX3, and a PX domain-containing protein; these pockets were disorganized in a EhVps26 truncated version lacking the IDR. The AlphaFold2 software predicted the interaction of EhVps26 with EhVps35, EhVps29 and EhSNX3, in a model similar to the reported mammalian crystals. By confocal and transmission electron microscopy, EhVps26 was found in the trophozoites plasma membrane, cytosol, endosomes, and Golgi-like apparatus. During phagocytosis, it followed the erythrocytes pathway, probably participating in cargoes selection and recycling. Ehvps26 gene knocking down evidenced that the EhVps26 protein is necessary for efficient phagocytosis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Martínez-Valencia 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.)

Background and Purpose: Leishmaniasis is a globally prevalent vector-borne disease caused by parasites of the genus Leishmania. The available chemotherapeutic drugs present problems related to efficacy, emergence of parasite resistance, toxicity and high cost, justifying the search for new drugs. Several classes of compounds have demonstrated activity against Leishmania, including icetexane-type diterpenes, previously isolated from Salvia and other Lamiaceae genera. Thus, in this study, compounds of Salvia procurrens were investigated for their leishmanicidal and immunomodulatory activities., Methods: The exudate of S. procurrens was obtained by rapidly dipping the aerial parts in dichloromethane. The compounds were isolated by column and centrifugal planar chromatography over silica gel. The effects on L. amazonensis growth, survival, membrane integrity, reactive oxygen species (ROS) generation, mitochondrial membrane potential and cytotoxicity of the compounds towards human erythrocytes, peripheral blood mononuclear cells and macrophages were evaluated. The effects on intracellular amastigote forms, nitric oxide (NO) and TNF-α production were also investigated., Results: The exudate from the leaves afforded the novel icetexane 7-hydroxyfruticulin A (1) as well as the known demethylisofruticulin A (2), fruticulin A (3) and demethylfruticulin A (4). The compounds (1-4) were tested against promastigotes of L. amazonensis and showed an effective inhibition of the parasite survival (IC 50 = 4.08-16.26 μM). In addition, they also induced mitochondrial ROS production, plasma membrane permeability and mitochondrial dysfunction in treated parasites, and presented low cytotoxicity against macrophages. Furthermore, all diterpenes tested reduced the number of parasites inside macrophages, by mechanisms involving TNF-α, NO and ROS., Conclusion: The results suggest the potential of 7-hydroxyfruticulin A (1) as well as the known demethylisofruticulin A (2),fruticulin A (3) and demethylfruticulin A (4) as candidates for use in further studies on the design of anti-leishmanial drugs., Competing Interests: Declaration of competing interest The authors declare that is no competing financial interest., (Copyright © 2024. Published by Elsevier GmbH.)

Background: Plasmodium falciparum, the malaria-causing parasite, is a leading cause of infection-induced deaths worldwide. The preferred treatment approach is artemisinin-based combination therapy, which couples fast-acting artemisinin derivatives with longer-acting drugs, such as lumefantrine, mefloquine, and amodiaquine. However, the urgency for new treatments has risen due to the parasite's growing resistance to existing therapies. In this study, a common characteristic of the P. falciparum proteome-stretches of poly-lysine residues, such as those found in proteins related to adhesion and pathogenicity-is investigated for its potential to treat infected erythrocytes., Methods: This study utilizes in vitro culturing of intra-erythrocytic P. falciparum to assess the ability of poly-lysine peptides to inhibit the parasite's growth, measured via flow cytometry of acridine orange-stained infected erythrocytes. The inhibitory effect of many poly-lysine lengths and modifications were tested this way. Affinity pull-downs and mass spectrometry were performed to identify the proteins interacting with these poly-lysines., Results: A single dose of these poly-basic peptides can successfully diminish parasitemia in human erythrocytes in vitro with minimal toxicity. The effectiveness of the treatment correlates with the length of the poly-lysine peptide, with 30 lysine peptides supporting the eradication of erythrocytic parasites within 72 h. PEG-ylation of the poly-lysine peptides or utilizing poly-lysine dendrimers and polymers retains or increases parasite clearance efficiency and bolsters the stability of these potential new therapeutics. Lastly, affinity pull-downs and mass-spectrometry identify P. falciparum's outer membrane proteins as likely targets for polybasic peptide medications., Conclusion: Since poly-lysine dendrimers are already FDA-approved for drug delivery and this study displays their potency against intraerythrocytic P. falciparum, their adaptation as anti-malarial drugs presents a promising new therapeutic strategy for malaria., (© 2024. The Author(s).)

For over a century, the need to identify malaria in the peripheral blood has been the driving force behind the development of fundamental clinical microscopy techniques. In the study by Moysis et al., artificial intelligence-based model was utilized to identify and provide quantitative morphological characteristics of red blood cells typical to severe malaria anaemia, irrespective to the actual presence of visible parasites. Commentary on: Moysis et al. Leveraging deep learning for detecting red blood cell morphological changes in blood films from children with severe malaria anaemia. Br J Haematol 2024;205:699-710., (© 2024 The Author(s). British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

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.)

Malaria continues to be a significant burden, particularly in Africa, which accounts for 95% of malaria deaths worldwide. Despite advances in malaria treatments, malaria eradication is hampered by insecticide and antimalarial drug resistance. Consequently, the need to discover new antimalarial lead compounds remains urgent. To help address this need, we evaluated the antiplasmodial activity of twenty-two amides and thioamides with pyridine cores and their non-pyridine analogues. Twelve of these compounds showed in vitro anti-proliferative activity against the intraerythrocytic stage of Plasmodium falciparum, the most virulent species of Plasmodium infecting humans. Thiopicolinamide 13i was found to possess submicromolar activity (IC 50  = 142 nM) and was >88-fold less active against a human cell line. The compound was equally effective against chloroquine-sensitive and -resistant parasites and did not inhibit β-hematin formation, pH regulation or PfATP4. Compound 13i may therefore possess a novel mechanism of action., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The virulence of parasites is expected to reflect an evolutionary tradeoff between increasing proliferation rates that enhance transmission and host mortality which curtails transmission. However, host resource availability may also limit parasites' proliferation rate. To understand the role of resource limitation as a driver of virulence evolution, Pak et al. (2024) use a within-host model of red blood cell (RBC) invasion by Plasmodium chabaudi. They find that within-host resource consumption limits the evolution of the parasite's proliferation rate, as the depletion of RBCs during infection results in intermediate optimal virulence. These results suggest that resource limitation, rather than host mortality, may drive the evolution of virulence., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Society for the Study of Evolution (SSE). All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Pathogens such as Plasmodium, Babesia, and Theileria invade and multiply within host red blood cells, leading to the pathological consequences of malaria, babesiosis, and theileriosis. Establishing continuous in vitro culture systems and suitable animal models is crucial for studying these pathogens. This review spotlights the Babesia duncani in culture-in mouse (ICIM) model as a promising resource for advancing research on the biology, pathogenicity, and virulence of intraerythrocytic parasites. The model offers practical benefits, encompassing well-defined culture conditions, ease of manipulation, and a well-annotated genome. Moreover, B. duncani serves as a surrogate system for drug discovery, facilitating the evaluation of new antiparasitic drugs in vitro and in animals, elucidating their modes of action, and uncovering potential resistance mechanisms. The B. duncani ICIM model thus emerges as a multifaceted tool with profound implications, promising advancements in our understanding of parasitic biology and shaping the development of future therapies., Competing Interests: Potential conflicts of interest. Both authors: No reported conflicts of interest. Both authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The currently accepted initiation of Babesia infection describes a sporozoite stage infused into the host, along with other saliva components, by the tick vector. This sporozoite can enter and initiate erythrocyte infection directly. In the particular case of Babesia microti , however, that sporozoite loses the ability to further propagate in vitro once deprived of its natural host. True B. sensu stricto do not require the host collaboration described in this study. Hence it has become a current topic of research involving B. microti ( B. sensu lato ), a rather unique species that requires host collaboration to maintain an erythrocyte propagation cycle. The main attachment protein is synthesized by this parasite in excess and exported to the host from the erythrocyte infrastructure to immunize the host at all stages of infection. The synthesis of host immune IgM antibody is necessary for the propagation of B. microti , being central to entry into uninfected host erythrocytes. Sequential use of the host immune system then involves complement factor C3b to complete the three-part assembly necessary to initiate the rhoptry sequence for invasion of uninfected erythrocytes and further propagation. These several components must be furnished within the in vitro culture medium and the sequence of these reactions is discussed. The corollary view of the parasite survival versus the host immune defenses is also discussed as it involves the same host factors promoting continuing parasite growth. This is the first description of continuous in vitro propagation of B. microti ., Competing Interests: The author declares no conflict of interest.

Strain-transcending antibodies against virulence-associated subsets of P. falciparum -infected erythrocyte surface antigens could protect children from severe malaria. However, the evidence supporting the existence of such antibodies is incomplete and inconsistent. One subset of surface antigens associated with severe malaria, rosette-mediating Plasmodium falciparum Erythrocyte Membrane Protein one (PfEMP1) variants, cause infected erythrocytes to bind to uninfected erythrocytes to form clusters of cells (rosettes) that contribute to microvascular obstruction and pathology. Here, we tested plasma from 80 individuals living in malaria-endemic regions for IgG recognition of the surface of four P. falciparum rosetting strains using flow cytometry. Broadly reactive plasma samples were then used in antibody elution experiments in which intact IgG was eluted from the surface of infected erythrocytes and transferred to heterologous rosetting strains to look for strain-transcending antibodies. We found that seroprevalence (percentage of positive plasma samples) against allopatric rosetting strains was high in adults (63%-93%) but lower in children (13%-48%). Strain-transcending antibodies were present in nine out of eleven eluted antibody experiments, with six of these recognizing multiple heterologous rosetting parasite strains. One eluate had rosette-disrupting activity against heterologous strains, suggesting PfEMP1 as the likely target of the strain-transcending antibodies. Naturally acquired strain-transcending antibodies to rosetting P. falciparum strains in humans have not been directly demonstrated previously. Their existence suggests that such antibodies could play a role in clinical protection and raises the possibility that conserved epitopes recognized by strain-transcending antibodies could be targeted therapeutically by monoclonal antibodies or vaccines., Competing Interests: The authors declare no conflict of interest.

Human babesiosis is a malaria-like illness caused by protozoan parasites of the genus Babesia . Babesia microti is responsible for most cases of human babesiosis in the United States, particularly in the Northeast and the Upper Midwest. Babesia microti is primarily transmitted to humans through the bite of infected deer ticks but also through the transfusion of blood components, particularly red blood cells. There is a high risk of severe and even fatal disease in immunocompromised patients. To date, serology testing relies on an indirect immunofluorescence assay that uses the whole Babesia microti antigen. Here, we report the construction of phage display cDNA libraries from Babesia microti -infected erythrocytes as well as human reticulocytes obtained from donors with hereditary hemochromatosis. Plasma samples were obtained from patients who were or had been infected with Babesia microti . The non-specific antibody reactivity of these plasma samples was minimized by pre-exposure to the human reticulocyte library. Using this novel experimental strategy, immunoreactive segments were identified in three Babesia microti antigens termed BmSA1 (also called BMN1-9; BmGPI12), BMN1-20 (BMN1-17; Bm32), and BM4.12 (N1-15). Moreover, our findings indicate that the major immunoreactive segment of BmSA1 does not overlap with the segment that mediates BmSA1 binding to mature erythrocytes. When used in combination, the three immunoreactive segments form the basis of a sensitive and comprehensive diagnostic immunoassay for human babesiosis, with implications for vaccine development., Competing Interests: The authors declare no conflict of interest.

The emergence of clinically drug-resistant malaria parasites requires the urgent development of new drugs. Mosquitoes are vectors of multiple pathogens and have developed resistance mechanisms against them, which often involve antimicrobial peptides (AMPs). An-cecB is an AMP of the malaria-transmitting mosquito genus Anopheles , and we herein report its antimalarial activity against Plasmodium falciparum 3D7, the artemisinin-resistant strain 803, and the chloroquine-resistant strain Dd2 in vitro . We also demonstrate its anti-parasite activity in vivo , using the rodent malaria parasite Plasmodium berghei (ANKA). We show that An-cecB displays potent antimalarial activity and that its mechanism of action may occur through direct killing of the parasite or through interaction with infected red blood cell membranes. Unfortunately, An-cecB was found to be cytotoxic to mammalian cells and had poor antimalarial activity in vivo . However, its truncated peptide An-cecB-1 retained most of its antimalarial activity and avoided its cytotoxicity in vitro . An-cecB-1 also showed better antimalarial activity in vivo . Mosquito-derived AMPs may provide new ideas for the development of antimalarial drugs against drug-resistant parasites, and An-cecB has potential use as a template for antimalarial peptides., Competing Interests: The authors declare no conflict of interest.

A hallmark of cerebral malaria (CM) is sequestration of Plasmodium falciparum -infected erythrocytes (IE) within the brain microvasculature. Binding of IE to endothelium reduces microvascular flow and, combined with an inflammatory response, perturbs endothelial barrier function, resulting in breakdown of the blood-brain barrier (BBB). Cytoadherence leads to activation of the endothelium and alters a range of cell processes affecting signaling pathways, receptor expression, coagulation, and disruption of BBB integrity. Here, we investigated whether CM-derived parasites elicit differential effects on human brain microvascular endothelial cells (HBMECs), as compared to uncomplicated malaria (UM)-derived parasites. Patient-derived IE from UM and CM clinical cases, as well as non-binding skeleton-binding protein 1 knockout parasites, were overlaid onto tumour necrosis factor (TNF)-activated HBMECs. Gene expression analysis of endothelial responses was performed using probe-based assays of a panel of genes involved in inflammation, apoptosis, endothelial barrier function, and prostacyclin synthesis pathway. We observed a significant effect on endothelial transcriptional responses in the presence of IE, yet there was no significant correlation between HBMEC responses and type of clinical syndrome (UM or CM). Furthermore, there was no correlation between HBMEC gene expression and both binding itself and level of IE binding to HBMECs, as we detected the same change in endothelial responses when employing both binding and non-binding parasites. Our results suggest that interaction of IE with endothelial cells in this co-culture model induces some endothelial responses that are independent of clinical origin and independent of the expression of the major variant antigen Plasmodium falciparum erythrocyte membrane protein 1 on the IE surface., Importance: Cerebral malaria (CM) is the most prevalent and deadly complication of severe Plasmodium falciparum infection. A hallmark of this disease is sequestration of P. falciparum -infected erythrocytes (IE) in brain microvasculature that ultimately results in breakdown of the blood-brain barrier. Here, we compared the effect of P. falciparum parasites derived from uncomplicated malaria (UM) and CM cases on the relative gene expression of human brain microvascular endothelial cells (HBMECs) for a panel of genes. We observed a significant effect on the endothelial transcriptional response in the presence of IE, yet there is no significant correlation between HBMEC responses and the type of clinical syndrome (UM or CM). Furthermore, there was no correlation between HBMEC gene expression and both binding itself and the level of IE binding to HBMECs. Our results suggest that interaction of IE with endothelial cells induces endothelial responses that are independent of clinical origin and not entirely driven by surface Plasmodium falciparum erythrocyte membrane protein 1 expression., Competing Interests: The authors declare no conflict of interest.

AbstractVector-borne blood parasites cause myriad sublethal effects and can even be deadly to endotherms, but far less is known about their impacts on ectothermic hosts. Moreover, the pathologies documented in endotherms are generally linked to infection by blood parasites rather than by their vectors. Here, we measured hematocrit, hemoglobin, and relative proportions of immature red blood cells to evaluate the physiological effects of two blood-feeding parasites and coinfection on ectothermic hosts, differentiating among pathological responses, extrinsic factors, and natural variations. We investigated a population of wild eastern hellbender salamanders ( Cryptobranchus alleganiensis ), which harbor leeches ( Placobdella appalachiensis ) that transmit blood parasites ( Trypanosoma spp.) to their hosts, often resulting in coinfection. We observed seasonal changes in host hematology corresponding to water temperature and demonstrated their ability to modulate hematological parameters in response to acute stress. We reveal seasonal relationships between parasite dynamics and host physiology, in which peak parasitemia occurred when hosts had seasonally high hematocrit and hemoglobin concentrations. We found that coinfected individuals expressed symptoms of anemia, including a regenerative response to depletion of their red blood cells. We also documented a more pronounced pathological response to leech vectors than to the trypanosomes they transmit. Our research underscores the complex interactions between host physiology, multiple parasites, and environmental factors and highlights the pathologies associated with the vector in coinfections. Given the contributions of climate change and disease in the rapid global decline of ectotherms such as amphibians, our study provides timely foundational insights into multiple factors that influence their red blood cell physiology.

For parasites, robust proliferation within hosts is crucial for establishing the infection and creating opportunities for onward transmission. While faster proliferation enhances transmission rates, it is often assumed to curtail transmission duration by killing the host (virulence), a trade-off constraining parasite evolution. Yet in many diseases, including malaria, the preponderance of infections with mild or absent symptoms suggests that host mortality is not a sufficient constraint, raising the question of what restrains evolution toward faster proliferation. In malaria infections, the maximum rate of proliferation is determined by the burst size, the number of daughter parasites produced per infected red blood cell. Larger burst sizes should expand the pool of infected red blood cells that can be used to produce the specialized transmission forms needed to infect mosquitoes. We use a within-host model parameterized for rodent malaria parasites (Plasmodium chabaudi) to project the transmission consequences of burst size, focusing on initial acute infection where resource limitation and risk of host mortality are greatest. We find that resource limitation restricts evolution toward higher burst sizes below the level predicted by host mortality alone. Our results suggest resource limitation could represent a more general constraint than virulence-transmission trade-offs, preventing evolution towards faster proliferation., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Society for the Study of Evolution (SSE).)