We developed inducible dead Cas12a (dCas12a)-based CRISPR interference/activation (CRISPRi/a) systems for Plasmodium falciparum, expanding the gene regulation toolkit beyond dCas9. Our previous systems fused dCas9 to the catalytic domains of PfSir2a or PfGCN5 under DiCre/loxP regulon, enabling conditional gene regulation but remaining constrained by Cas9's NGG protospacer adjacent motif (PAM) requirement. Unlike dCas9, dCas12a recognizes T-rich PAMs and processes a single transcript into multiple sgRNAs via its RNase activity, allowing multiplexed regulation of one or multiple genes. Functional validation targeting EBA175 and ATG18 demonstrated that single sgRNAs mediated CRISPRi/a's active gene regulation, while multiplexed sgRNA arrays enhanced CRISPRi activities. Using this platform, we showed that conditional repression of the histone methyltransferase gene PfSET7 impairs schizont egress and merozoite invasion, highlighting its critical role in parasite multiplication, consistent with its Toxoplasma homolog TgADTM1. Altogether, these results establish dCas12a-based CRISPRi/a as a versatile platform for conditional and multiplexed gene regulation in P. falciparum.

The malaria parasite, Plasmodium falciparum, replicates asexually within red blood cells by schizogony, a mode of division in which organelles divide multiple times before a final coordinated cytokinesis event, called segmentation, partitions them into dozens of merozoites. The Plasmodium cytoskeleton functions critically during this process, facilitating one-per-cell organelle segregation. Here, we identify and characterize a cytoskeleton-associated protein, PfOGRE (organelle retention, PF3D7_1435600), required for segregation of the mitochondrion and apicoplast, a non-photosynthetic plastid. Immunofluorescence paired with ultrastructure expansion microscopy demonstrates that PfOGRE localizes alongside subpellicular microtubules in nascent merozoites, adjacent to the segregating apicoplast. Conditional knockout of PfOGRE produces organelle-less merozoites with abnormal subpellicular microtubules, impairing asexual replication. Immunoprecipitation and mass spectrometry identify several candidate interactors, including PfMyoF. Conditional knockdown of PfMyoF produces similar organelle segregation defects. Together, these results establish PfOGRE as a critical mediator of organelle segregation and define a non-structural role for Plasmodium subpellicular microtubules during this process.

Plasmodium parasites rely on diverse protein kinases to regulate critical processes, yet many remain uncharacterised. Protein kinase 2 (PK2) is required for asexual blood-stage proliferation and merozoite invasion, but its role in sexual stages and transmission is unknown. Here, using live-cell and super-resolution imaging, ultrastructural analysis, functional phenotyping, and quantitative phosphoproteomics, we define the role of PK2 in Plasmodium berghei transmission. PK2 was expressed in merozoites, ookinetes, and sporozoites: the three invasive forms of the parasite. Conditional depletion of PK2 impaired ookinete motility, disrupted apical microneme positioning, abolished oocyst formation, and prevented mosquito-to-host transmission. Direct haemocoel injection of mutant ookinetes failed to restore sporozoite production, demonstrating an additional requirement for PK2 downstream of midgut traversal. Although global protein abundance was largely preserved, phosphoproteomic profiling identified altered phosphorylation of proteins associated with cytoskeletal organisation, host-cell interaction, midgut invasion, and gene regulation. These findings highlight PK2's critical role across multiple invasive stages, advancing understanding of malaria parasite transmission and identifying putative PK2-regulated pathways as potential targets for transmission-blocking interventions.

Cerebral malaria involves the expression of a particular var subgroup, termed upsA, in Plasmodium falciparum, which encodes a subset of PfEMP1 variants that avidly bind to human receptors on endothelial cells, resulting in obstruction of microvasculature in the brain and severe pathogenesis. Despite the clinical importance of the upsA var genes, the mechanism of their low-frequency activation in asymptomatic or uncomplicated malaria cases remains poorly understood. Here, we identify a JmjC-domain-containing histone demethylase PfJmjC1 as a vital transcriptional silencer of upsA var genes, which may negatively regulate malaria pathogenesis. Genome-wide profiling reveals that PfJmjC1 localizes to heterochromatic loci including the entire var gene family, and interacts with canonical heterochromatin factors such as HP1 at the schizont stage. However, PfJmjC1 preferentially binds to the promoter regions of upsA var loci in ring-stage parasites in which non-upsA vars are activated. Genetic deletion or conditional knockdown of PfJmjC1 selectively derepresses upsA var and disrupts the var expression "counting" mechanism, accompanied by increased H3K4me3 and H3K9ac, as well as decreased H3K9me3 modification at upsA var loci. The altered PfEMP1 expression patterns and acquisition of multiple endothelial receptor-binding phenotypes are experimentally verified at the single-cell level. Additionally, Hi-C-seq and three-dimensional genome modeling show that PfJmjC1 deficiency reorganizes nuclear heterochromatin architecture, repositioning upsA var loci toward a euchromatic environment. Together, these findings not only uncover a mechanism controlling the transcriptional activity of severe malaria-associated virulence genes but also provide a potential therapeutic target against severe malaria.

Finished herbal antimalarials are widely used in Ghana, although scientific reports regarding their antiplasmodial activity and safety of these products are limited. This study, therefore, assessed the antiplasmodial activities and safety of three widely distributed antimalarial polyherbal products. These products (samples A, B and C) were obtained through a minisurvey conducted in the Accra and Kumasi Metropolises. The SYBR Green fluorescence antiplasmodial assay was conducted on different stages of chloroquine-resistant and chloroquine-sensitive strains of P. falciparum. Cytotoxicity and subacute toxicity were assessed following MTT assay and OECD guidelines, respectively. Samples A, B and C yielded IC50 (μg/mL) values of 0.869 ± 0.10, 0.828 ± 0.05 and 3.07 ± 0.098, respectively, against the chloroquine-sensitive (3D7) strain at the trophozoite stage. At the schizont stage, samples A, B and C exhibited moderate to weak activities with IC50 (μg/mL) values of 9.96 ± 0.63, 7.71 ± 1.63 and 13.66 ± 1.91, respectively. At the gametocyte stage, samples A, B and C displayed moderate to weak activity with IC50 (μg/mL) values of 13.59 ± 0.49, 17.92 ± 0.814 and 59.65 ± 1.61, respectively. Parasite inhibition against the chloroquine-resistant strain (Dd2) at the trophozoite stage demonstrated good antiplasmodial activities for samples A and B, with IC50 (μg/mL) values of 0.939 ± 0.04 and 0.318 ± 0.01, respectively. Sample C yielded an IC50 (μg/mL) value of 15.09 ± 0.319. In conclusion, the three products are antiplasmodial agents and are safe in the rodent model.

Theileria annulata, an apicomplexan parasite, causes lymphoproliferative disease in cattle and threatens the bovine industry in tropical and subtropical regions. To clarify life-cycle mechanisms and parasite-host interactions, we conducted high-resolution Orbitrap proteomic and phosphoproteomic analyses of the schizont and piroplasm stages. In total, we confidently identified and quantified 2,093 proteins; among these, 459 proteins were observed in a phosphorylated state, comprising 5,069 distinct phosphorylation sites. Functional enrichment analysis of differentially abundant proteins indicated stage-associated remodeling of genetic information processing, carbohydrate, lipid, and amino-acid metabolism, and core cellular processes. Stage-associated proteins were linked to host-parasite interaction, mitochondrial function, and metabolic adaptation. Schizonts showed broader proteomic and phosphorylation profiles consistent with higher biosynthetic and energy demands. In contrast, piroplasms exhibited a more restricted proteomic profile associated with erythrocyte adaptation and invasion-related functions. Together, these stage-resolved datasets provide a reference for proteome and phosphoproteome remodeling across development and prioritize candidate stage-associated proteins and phosphosites for future functional validation.

Eimeria gilruthi, an underreported coccidian parasite, primarily infects the abomasum of small ruminants. It has an unclear life cycle, and its phylogenetic relationship remains poorly understood. Most E. gilruthi infections are incidental, with rare descriptions of those associated with clinical disease. This study aims to characterize the lesions, immune sresponse, and molecular identity of E. gilruthi in naturally infected small ruminants. Histopathology, special stains, and immunohistochemistry for immune cells were used to assess tissue responses in the abomasum of 4 goats and one sheep. Molecular characterization was performed targeting the 18S and ITS1 regions of its rDNA and cox3 regions of its mDNA, followed by phylogenetic analysis. All animals harbored megaloschizonts within hypertrophic host cells, accompanied by lymphohistiocytic and eosinophilic abomasitis in the deep mucosa without evidence of sexual stages or oocyst formation. Immunohistochemistry revealed an inflammatory infiltrate composed of CD3+ T and CD20+ B lymphocytes and IBA-1+ macrophages, especially around immature schizonts, with fewer eosinophils. Molecular analysis revealed genetic divergence among isolates. While Eimeria from goats clustered with E. gilruthi, E. christenseni, and E. alpacae, Eimeria from sheep grouped with E. bovis, E. zuernii, and E. ellipsoidalis, suggesting potential host-specific lineages or different species. These findings enhance current understanding of E. gilruthi immune response and emphasize the need for further research into its taxonomy, life cycle, and host specificity to clarify its impact on small ruminant health.

Malaria parasites must undergo complex developmental transitions to complete their life cycle and transmit between vertebrate and mosquito hosts. These transitions are tightly regulated by protein phosphorylation events, yet the specific kinases involved remain poorly characterized. Here, we investigate the role of a previously uncharacterized tyrosine kinase-like protein, TKL3, in the rodent malaria parasite Plasmodium berghei. We show that TKL3 is expressed in blood stages, schizonts, gametocytes, zygotes, and ookinetes and localizes to the cytoplasm. Targeted disruption of the TKL3 gene impairs asexual blood-stage growth, male gamete fertility, and consequently compromises ookinete development. Although oocyst numbers are significantly reduced in mosquitoes, the sporozoites that do form are morphologically normal, retain hepatocyte infectivity, and complete liver-stage development. However, TKL3 knockout sporozoites showed delayed blood-stage patency due to reduced asexual replication. These findings identify TKL3 as an important regulator of parasite growth and transmission, providing new insights into kinases involved in the Plasmodium life cycle.

Natural, non-genetic variation among host cells can shape infection outcomes, yet its role in intracellular parasite dynamics remains poorly understood. Here, we show that variability in hemoglobin (Hb) content among individual red blood cells (RBCs) modulates nutrient consumption by Plasmodium falciparum, the most lethal malaria parasite. Using label-free quantitative-phase imaging (QPI), we measured cytosolic Hb in thousands of uninfected and infected RBCs across the 48-h asexual cycle. Hb content varied widely among uninfected cells, and after invasion, infected cells mirrored and amplified this distribution: variability increased from 17% in uninfected RBCs to >100% in schizonts. A minimal predator-prey model accurately reproduced the observed spread in parasite Hb consumption, showing that host variability alone can generate much of the diversity in parasite feeding rates traditionally attributed to parasite factors. These findings provide the first evidence that host phenotypic noise drives variation in Hb consumption among genetically identical parasites. Incorporating host-to-host variability into within-host models could improve predictions of parasite growth, refine the activation kinetics of Hb-dependent antimalarials, and reveal analogous principles in other intracellular infections where host variability and host-parasite crosstalk can shape pathogen outcomes. This systems-level perspective emphasizes the need to account for host heterogeneity in both experimental design and therapeutic strategies.IMPORTANCECell-to-cell phenotypic variability is a fundamental feature of living systems, yet its impact on host-pathogen interactions has been largely overlooked. Here, we show that natural, non-genetic variation in red blood cell hemoglobin strongly shapes how Plasmodium falciparum-the most lethal malaria parasite-consumes Hb inside host cells. Using quantitative imaging and a mathematical model, we demonstrate that host variability alone can explain much of the observed differences in parasite feeding rates. This perspective reframes parasite Hb consumption as an emergent property of host-parasite interactions. More broadly, it highlights how natural variability among host cells can influence the course of intracellular infections, with potential implications for predicting pathogen growth and optimizing drug treatment strategies.

Eimeria bovis is an obligate intracellular apicomplexan parasite and the major agent of bovine coccidiosis. Intracellular E. bovis first merogony results in the formation of large macromeronts (≤ 400 µm) containing up to 140,000 merozoites I. This extensive replication imposes substantial metabolic stress on bovine endothelial host cells, leading to mitochondrial dysfunction and premature senescence. During macromeront development, non-infected bystander cells (BCs) accumulate around infected host cells and previous findings provided evidence for tunneling nanotube (TNT)-based interactions between infected cells and BCs. In the current study, we aimed to further analyze interactions between infected cells and BCs, potentially forming part of a multicellular support mechanism promoting macromeront development. To dissect BC-based reactions, we used a meront-transfer-system, which allowed for selective treatments of non-infected cells, serving as BCs in mixed cultures with E. bovis meront-carrying host cells (MCHCs). When addressing gap junction formation, protein expression profiles of E. bovis-infected cell layers revealed a significant upregulation of the connexins Cx37 and Cx40, but not of Cx43 during macromeront formation. In addition, E. bovis infection altered the subcellular distribution of Cx37, Cx40 and Cx43. The functional relevance of connexins was supported by blocking gap junction formation in BCs with carbenoxolone or Gap26 (inhibits Cx37, Cx40 and Cx43), both of which impaired parasite development. Besides connexins, we also studied TNT-based BC-MCHC-interactions in more detail. When quantitatively estimating BC-mediated donation of mitochondria to E. bovis-infected cells to improve MCHC bioenergetics, we showed that a high proportion of MCHCs (89%) obtained mitochondria from BCs. In line, the disruption of microtubule integrity by nocodazole treatments in BCs diminished both TNT formation and merozoite I production. Moreover, inhibitor-based impairment of BC metabolism demonstrated that intact glycolytic and glutaminolytic activities in addition to functional cholesterol esterification and trafficking are required in BCs to sustain effective merozoite I production in BC-MCHC-cultures. Collectively, the current findings support the hypothesis that effective intracellular E. bovis macromeront formation depends on an environmental BC-based support network involving BC metabolic integrity, short-range gap-junctional small molecule transfer, and long-range TNT-mediated mitochondria donation.

The protozoan parasite Perkinsus olseni actively proliferates within marine molluscan hosts and causes serious negative effects on mollusk production worldwide. Recent research revealed that one Japanese isolate requires exogenous glycine betaine (GB) for vegetative proliferation, whereas isolates from some congeneric species do not, suggesting that this requirement is a derived characteristic of this genus. In the present study, we established various isolates from Japan, Europe, and Oceania, and showed that P. olseni isolates from Japan and Europe commonly require exogenous GB, whereas Oceanian isolates do not. This suggests that Oceanian isolates may retain ancestral-like characteristics compared to Japanese and European isolates. Analyses of the non-transcribed spacer region of ribosomal DNA further support the above interpretation that the Oceanian isolates represent a more basal split, and additionally suggest that the Japanese and European isolates belong to lineages that have undergone relatively recent geographic expansion. Morphological comparisons among isolates showed that the Oceanian isolates possessed larger schizonts and trophozoites, producing fewer daughter cells, but formed larger hypnospores capable of producing a greater number of zoospores, although the association between GB requirements and morphological differences remains unknown. The present study demonstrates the existence of distinct lineages of this parasite and provides novel insights into the evolution, geographical dispersion, and shifts in life-history strategies of P. olseni.

The recent rise in malaria cases post-COVID-19 and antimalarial resistance in Plasmodium falciparum present new treatment challenges. Repurposing existing drugs or creating new combinations is an effective strategy. Elemental zinc (Zn[2+]) mitigates cancer progression and viral infections. However, no study has demonstrated the direct lethal effect of zinc on blood-stage P. falciparum. This study examined Zn[2+] antiplasmodial effect on the blood-stage P. falciparum 3D7 strain using a time-inhibition growth assay (24, 48, 72, and 96 h) with the SYBR Green I method. The findings indicated that Zn[2+] exhibited IC50 values of 49.5 and 26 µM at 72 and 96 h, respectively. When fixed Zn[2+] concentrations (50 and 25 µM) were co-treated for 48 h with standard antimalarial drugs, such as chloroquine (CQ) and dihydroartemisinin (DHA), there was a notable decrease in CQ IC50 value (0.017 and 0.026 µM) and DHA IC50 value (0.007 and 0.011 µM) compared to treatment with these drugs alone. Stage-specific studies revealed that trophozoites and schizonts were highly susceptible to Zn[2+] in combination with CQ or DHA. This study is the first to show that elemental Zn[2+] alone impedes blood-stage parasites in a time- and stage-dependent manner and its combination with CQ or DHA improves antimalarial treatment effectiveness.

Plasmodium cynomolgi is one of the few malaria parasite species that forms dormant liver stages known as hypnozoites and is therefore a suitable model for Plasmodium vivax, the causative agent of recurrent malaria. Very little is known about the biology and pharmacology of liver stage dormancy, which hampers the search for compounds with anti-hypnozoite activity.