Plasmodium falciparum employs sophisticated strategies to subvert host immunity, whereas the specific parasite-derived factors governing this critical process remain elusive. Here, by utilizing P. falciparum 3D7 and P. berghei ANKA infection models, we investigated the role of parasite-derived PI3K in immune evasion, and found that Plasmodium PI3K prevents the externalization of phosphatidylserine (PS), a canonical prophagocytic ‘eat-me’ signal. Inhibiting PI3K activity in infected red blood cells (iRBCs) significantly increased M2 macrophage polarization and iRBC recognition. Mechanistically, we identified that PfPI3K preserves mitochondrial membrane potential and prevents calcium efflux by promoting the 2-hydroxyisobutyrylation of the 14–3-3 protein to maintain a calcium-homeostatic environment. Therefore, PfPI3K could phosphorylate P. falciparum phospholipid scramblase 1 (PfPLSCR1) to maintain PS internalization in iRBCs while preventing their exposure to immune cells. Collectively, these results revealed a mechanism through which Plasmodium parasites leverage PI3K to actively suppress PS-associated ‘eat-me’ signalling in iRBCs, thereby circumventing host macrophage-mediated surveillance and facilitating persistent infection.

The pathogenesis of Plasmodium falciparum malaria involves coordinated molecular events, including host cell invasion, intraerythrocytic replication cycles, and antigenic variation, which are critically dependent on the stage-specific regulation of parasite proteins. Despite their biological significance, the molecular governance of these pathogenic mechanisms remains inadequately characterized. Our investigation reveals that the expression dynamics of an ApiAP2 family transcription factor, PfAP2-V (PF3D7_1239200), exhibit strong temporal coordination with the transcriptional activation of virulence-associated genes during the blood-stage development of P. falciparum parasites. These genes encompass those coding for merozoite surface antigens and the inner membrane complex families and the var gene clusters encoding P. falciparum erythrocyte membrane protein 1 (PfEMP1) cytoadherence proteins. Conditional knockdown of pfap2-v expression significantly decreased parasite intraerythrocytic proliferation, resulted in structural abnormalities in infected erythrocyte surface topology, and markedly reduced the cytoadhesion capacity of infected erythrocytes to human endothelial receptors, such as CD36, ICAM-1, and PECAM-1/CD31. Mechanistic analyses revealed that PfAP2-V directly associates with specific chromosomal regions, where it is involved in chromatin interactions and facilitates transcriptional activation. These findings establish PfAP2-V as a master transcriptional regulator that governs both parasite proliferation and the expression of the virulence factors critical for P. falciparum malaria pathogenesis.

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. Severe malaria is linked to upsA var gene expression, whose regulation remains unclear. Here, the authors identify JmjC1 as an epigenetic regulator of upsA var genes at the transcriptional level through chromatin and nuclear organization.

Pazicky et al. introduced a thermal profiling method, meltome-assisted profiling of protein complexes (MAP-X), combined with machine learning, to map protein complex dynamics in intact Plasmodium falciparum, a deadly malaria parasite. This in situ approach reveals a highly dynamic interactome, identifying novel complexes and ‘moonlighting’ proteins, shifting our understanding from static lists to temporal networks.

ABSTRACT Regulatory B cells (Bregs) are a functionally defined yet phenotypically heterogeneous subset of lymphocytes that are essential for maintaining immune homeostasis. Their canonical function is regulated through the secretion of interleukin‐10 (IL‐10), a potent anti‐inflammatory cytokine. However, accumulating evidence indicates that other molecules, such as IL‐35 and transforming growth factor‐β, and that of contact‐dependent pathways, such as Programmed Cell Death Ligand‐1 (PD‐L1) and Programmed Cell Death‐1 (PD‐1), also play indispensable roles in their regulatory arsenal. This review examines the immunoregulatory roles of Bregs across diverse clinical contexts, including infectious diseases, cancers, autoimmune disorders (such as systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and uveitis), and organ transplantation. Crucially, we highlight a fundamental functional dichotomy: although Bregs confer protection against autoimmunity and promote transplant tolerance, they concurrently drive the progression of chronic infections and malignancies by dampening antipathogen and antitumor immune responses. This functional dichotomy highlights the complexity of immune regulation, where Bregs act as critical nodes balancing health and pathology. The immense therapeutic potential by modulating Bregs activity and unresolved questions that will guide the future frontiers of Bregs research are essentially discussed.

Apicomplexan protozoans employ an intricate invasion mechanism involving dynamic interactions with host cells, characterized by sequential secretion of adhesins and lectins. Our laboratory previously identified TgSABP1, a novel Toxoplasma gondii adhesin, demonstrating specific binding affinity for sialic acid (SA) receptors on host cell surfaces. However, the structural determinants governing SA recognition by this adhesin remain undefined. Three-dimensional structural predictions of TgSABP1 and homologous proteins were generated using AlphaFold2. Bio-layer interferometry (BLI) quantified the binding affinities between the recombinant proteins and ligands. Competitive BLI assays evaluated small molecules that potentially inhibit the TgSABP1–sialyllactose interactions. Molecular docking simulations employing AutoDock Vina software elucidated ligand-binding site interactions. In vitro invasion inhibition assays were performed to assess the therapeutic potential of lead compounds targeting TgSABP1 against T. gondii tachyzoites. AlphaFold2 structural predictions revealed that TgSABP1 and its homologues contain a conserved globular domain (pLDDT > 90) with significant structural homology (with root-mean-square deviation [RMSD] < 4 Å) to a Plasmodium falciparum invasion-related protein PfIMP2 (PDB: 5LG9). BLI quantification demonstrated the micromolar binding affinities of the recombinant proteins for 3′-sialyllactose-polyacrylamide (PAA) and 6′-sialyllactose (6′SL)-PAA. Intriguingly, although recombinant TgSABP1 showed stronger lactose binding (KD = 0.02 ± 0.01 M) compared to SA (KD = 2.07 ± 0.45 M), only the latter exhibited an inhibition on the TgSABP1-6′SL-PAA interaction. Virtual screening of Food and Drug Administration (FDA)-approved compounds identified eltrombopag as a high-affinity molecule (ΔGbind = −8.3 kcal/mol) targeting the SA-binding pocket in TgSABP1. Functional validation demonstrated that eltrombopag effectively blocked the TgSABP1/6′SL-PAA interaction and significantly decreased host cell invasion of T. gondii tachyzoites. Our study reveals a conserved globular domain of apicomplexan parasites as a novel SA-binding domain. Structural and functional characterization demonstrates its critical role in mediating TgSABP1-host cell interactions. Targeting this SA-binding pocket with eltrombopag effectively decreased T. gondii tachyzoite invasion, suggesting its therapeutic potential as an anti-invasion target. These findings not only elucidate a conserved mechanism underlying host receptor recognition in apicomplexans, but also establish a structural framework for the rational design of broad-spectrum inhibitors targeting invasion-related lectin domains.

Protein acetylation regulates essential processes across eukaryotes. In trypanosomatids, stage-specific acetylation suggests roles in parasite differentiation. Here, we functionally characterized zinc-dependent lysine deacetylases (DAC1, DAC3, DAC4, and DAC5) in Leishmania mexicana. CRISPR-Cas9-mediated disruption revealed that DAC1 and DAC3 are essential for procyclics, while DAC4 and DAC5 are dispensable. DAC1 and DAC5 are localized in the cytoplasm, and DAC3 and DAC4 in the nucleus. Functional analysis implicates DAC1, DAC3, and DAC5 in procyclic proliferation, whereas DAC1 and DAC5 drive promastigote-to-metacyclic differentiation. DAC5 was required for metacyclogenesis in the sand flies, the promastigote–amastigote transition, and amastigote intracellular replication. Notably, DAC5-null parasites failed to induce lesions in mice, displaying an attenuated phenotype. Proteomic profiling uncovered altered acetylation patterns in DAC mutants, linking DAC5 to cytoskeleton regulation and cell cycle control. These findings identify acetylation as a central regulator of Leishmania stage differentiation and highlight DAC5 as a key factor in parasite virulence.

Environmental factors restrict malaria parasite development, but the influence of host metabolic variations on the infectivity of the blood stage parasite is not fully understood. Here we show that mice on a ketogenic diet are completely protected from infection with the malaria parasite Plasmodium berghei. We further show that administration of the ketone body β-hydroxybutyrate (βOHB), but not of acetoacetate, increases survival of infected mice and inhibits proliferation of both P. berghei and Plasmodium falciparum in vitro. Administration of either a ketogenic diet or βOHB induces metabolic reprogramming in parasites, including reduced levels of nicotinamide adenine dinucleotide, which is associated with the downregulation of genes controlling parasite development, erythrocyte invasion and pathogenicity. Our data indicate that a ketogenic diet and the ketone body βOHB confer resistance to malaria in mice by causing developmental arrest of Plasmodium parasites, highlighting the potential of dietary and metabolic strategies to fight malarial infection. A ketogenic diet and the ketone body β-hydroxybutyrate are shown to confer resistance to malaria in mice by inducing metabolic reprogramming in Plasmodium parasites, suggesting a dietary strategy for malaria prevention.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite. Currently, the effective drugs for the treatment of toxoplasmosis are mainly pyrimethamine and sulfonamide, but these drugs have high toxicity and side effects, so the search for new and effective drug targets is urgent. Posttranslational modifications (PTMs) are certain chemical groups that are covalently coupled to specific amino acids within a protein. Studies have shown that T. gondii expresses a variety of proteins that require PTMs to regulate the parasite’s response to extracellular stimuli and life cycle transitions at different developmental stages. In this review, we summarize and analyze the 14 PTMs that have been found in T. gondii proteins to date and their roles in T. gondii growth and development. In addition, we discuss the potential crosstalk between T. gondii PTMs at different stages and summarize the results of studies on inhibitors that target PTM regulatory factors. The aim is to further analyze the functions of PTMs in the development and pathogenesis of T. gondii and to lay the foundation for the search for new anti‐ T. gondii drug targets.

BACKGROUND:To achieve sustainable and integrated control of schistosomiasis, it necessitates the implementation of comprehensive strategies, where effective vaccines could play a pivotal role. The limited identification and validation of schistosome antigens hinders the progress of vaccine development for the disease. Schistosome cysteine proteinases are considered as important targets for novel anti-schistosomiasis immunoprophylaxis due to their primary role in nutrient absorption. Previous research on the Schistosoma japonicum degradome has identified a group of cathepsin L-like proteases (SjCLs) that are up-regulated in hepatic schistosomula and adult worms. METHODS/FINDINGS:In this study, five recombinant proteins representing the mature form of these SjCLs, designated as rSjCL1-5, were successfully produced. Mice immunized with the rSjCLs were subsequently challenged with cercariae to evaluate the immunoprotective efficacy of these proteins. The expression and localization of SjCL1 were analyzed by qRT-PCR, western blotting and immunofluorescence assays. Among these five rSjCLs, only the immunization with rSjCL1 conferred partial protection to the mice against S. japonicum infection, resulting in a reduction in worm burden by 34.9% ~ 38.0% and a decrease of egg burden by 46.2% ~ 48.3%. This immunization also effectively mitigated body weight loss and hepatomegaly in the challenged mice. SjCL1 was primarily localized along the intestinal intima of hepatic schistosomula, as well as male and female adults, and on the tegument of male adults. The mature form of SjCL1 was detected in the excretory/secretory products of the parasites. Hepatic schistosomulum treated with SjCL1 antibodies in vitro showed significant growth retardation, although remained viable and developed intestinal heme pigmentation, indicative of hemoglobin digestion. CONCLUSIONS/SIGNIFICANCE:Our study revealed that SjCL1 is essential for normal parasite growth and shed new light for the development of schistosomiasis vaccines targeting cathepsins, which play a key role in the early intra-mammalian stages of schistosomes.

Tripartite motif-containing proteins (TRIMs), comprising the greatest subfamily of E3 ubiquitin ligases with approximately 80 members of this family, are widely distributed in mammalian cells. TRIMs actively participate in ubiquitination of target proteins, a type of post-translational modification associated with protein degradation and other functions. Tripartite motif-containing protein 29 (TRIM29), a member of the TRIM family, differs from other members of this family in that it lacks the RING finger structural domain containing cysteine and histidine residues that mediates DNA binding, protein-protein interactions, and ubiquitin ligase, at its N-terminus. The expression of TRIM29 was initially found to be associated with cancer and diabetic nephropathy progression, and antiviral immunity which is triggered by virus-derived nucleic acids binding to pattern recognition receptors (PRRs) on immune cells. Recently, TRIM29 has also been explored as a diagnostic biomarker and therapeutic target for some immune-related diseases. Here, we review the functions of TRIM29 in the progression of diseases and the inherent mechanisms, as well as the remaining gaps in the literature. A thorough understanding of the detailed regulatory mechanisms of TRIM29 will ultimately facilitate the development of different therapeutic strategies for various diseases.

Malaria, caused by Plasmodium parasites and transmitted by Anopheles mosquitoes, greatly impacts public health and socioeconomic development, particularly in sub-Saharan African countries. Despite advances in malaria treatment and prevention, the number of clinical cases and deaths have increased in recent years. The complex life cycle and genetic diversity of Plasmodium parasites pose significant challenges in drug and vaccine development, particularly due to the emerging partial resistance of parasites to artemisinin. With the availability and application of state-of-the-art biotechnology in recent years, knowledge in terms of parasite biology, pathogenicity, host-parasite interactions and pathogenesis has advanced tremendously. This review highlights the most recent research progress and understanding in Plasmodium biology, with a primary focus on P. falciparum and associated pathogenesis. The therapeutic targets and progress in the clinical application of anti-malaria drugs have also been summarized. The FDA-approved regimens like Artemether-Lumefantrine, Atovaquone-Proguanil, and Primaquine are discussed, and their benefits and limitations are highlighted, especially in terms of drug resistance. Perspectives in the development of novel vaccines and new drugs, such as Sevuparin, Imatinib, and Cipargamin, and combination therapies with promise in overcoming resistance has been proposed. Overall, this review provides a detailed summary of the latest progress in malaria research and emphasizes the need for continuous monitoring and innovation in malaria treatment.

Toxoplasma gondii infects nucleated cells of warm-blooded animals and cause zoonotic toxoplasmosis. Lysine lactylation, as a novel post-translational modification, is essential for epigenetic regulation and cellular processes, and proteomic analyses have shown that lactylated proteins are involved in a wide range of biological processes including energy metabolism, gene regulation, and protein biosynthesis. Additionally, protein lactylation is prevalent in T. gondii, while its regulatory mechanisms have not been fully understood. In this study, we investigated the role of T. gondii phosphofructokinase-2 (TgPFKII) and the adenosine-5'-monophosphate-activated protein kinase (AMPK) signaling pathway in the invasion, replication, and lactylation regulation of T. gondii. We localized TgPFKII in the cytoplasm of T. gondii tachyzoites and demonstrated its necessity for parasite growth and protein lactylation through auxin-induced degradation. Our results showed that inhibition of the AMPK pathway led to decreased TgPFKII expression and reduced protein lactylation levels. Furthermore, AMPK-specific inhibitors significantly impaired parasite invasion and proliferation. These findings highlight TgPFKII as a crucial regulator of lactylation and underscore the importance of the AMPK pathway in T. gondii's pathogenic mechanisms, offering potential targets for therapeutic intervention.IMPORTANCEUnderstanding the intricate mechanisms by which Toxoplasma gondii invades and proliferates within host cells is essential for developing novel therapeutic strategies against toxoplasmosis. This study focuses on the pivotal roles of T. gondii phosphofructokinase-2 (TgPFKII) and the adenosine-5'-monophosphate-activated protein kinase (AMPK) signaling pathway in regulating protein lactylation in association with parasite invasion and growth. By elucidating the cellular localization and functional importance of TgPFKII, as well as its regulation through AMPK-specific inhibitors, we provide comprehensive insights into the metabolic and signaling networks that underpin T. gondii pathogenicity. Our findings reveal that TgPFKII is a critical regulator of lactylation and that the AMPK pathway significantly influences T. gondii's ability to invade and replicate within host cells. These insights pave the way for targeted interventions aimed at disrupting key metabolic and signaling pathways in T. gondii, potentially leading to more effective treatments for toxoplasmosis.

Hypothesis: Trypanosomiasis is one of the main threats to human and animal health in African countries. Trypanosoma brucei can evade the host immune recognition by rapidly altering its variant surface glycoprotein (VSG). The ATP synthase F1 1 subunit of the parasite exhibits extremely low similarity to that of its mammalian hosts, hypothetically making it an ideal target for the development of novel therapeutics. Experiments: Graphene quantum dots (GQDs) were synthesized, and their adhesion to T. brucei surface and internalization was observed microscopically. The activity of ATP synthase and mitochondrial membrane potential of T. brucei were measured after exposure to GQDs. Proteomics, biolayer interferometry, and molecular dynamic simulations were utilized to evaluate the interaction between GQDs with the target proteins. Findings: GQDs specifically adhered to the VSG of T. brucei and were conveyed inside the parasite via the VSG internalization pathway. The GQDs promoted intracellular ROS production, interacted with, and inhibited the activity of the p18 subunit of ATP synthase, disrupted parasite mitochondrial membrane potential. Additionally, the GQDs caused a decrease in aminoacyl - tRNA biosynthesis, and upregulated RNA and protein degradation pathways. The findings of this study offer a novel avenue for the target-oriented discovery of anti-trypanosome drugs.

Parasitic diseases, caused by a diverse array of parasites, remain a substantial threat to global health. Toll-like receptor 3 (TLR3) represents a pivotal element in the innate immune system, distinguished by an ability to signal via the TIR-domain-containing adapter-inducing interferon-β (TRIF)-dependent pathway upon detecting pathogen-derived double-stranded RNA (dsRNA), exosomal RNA (exoRNA), and long non-coding RNA (lncRNA). Predominantly localized on endosomal membranes, TLR3 is extensively expressed in neurons, immune cells, fibroblasts, and epithelial cells. Upon activation, TLR3 engages adaptor molecules such as TRIF, facilitating the phosphorylation of TANK-binding kinase 1 and the subsequent activation of interferon regulatory factors. This signaling cascade triggers the production of type I interferons (IFN-α/β) and proinflammatory cytokines such as interleukin (IL)-6, IL-8, IL-12, and tumor necrosis factor-alpha, which are crucial for effective immune defense against infections. Recent findings highlight the essential role of TLR3 in parasitic infections by detecting nucleic acids from damaged cells to activate dendritic and natural killer cells. TLR3 also functions with other receptors, such as TLR2 and TLR4, to enhance cytokine production and improve parasite clearance. However, TLR3 overactivation can induce excessive, harmful inflammation and tissue damage, highlighting its dual role in balancing immune defense. This review comprehensively examines the TLR3 signaling pathway and its multifaceted role in various parasitic infections, including those caused by Plasmodium spp., Leishmania spp., Clonorchis sinensis, Schistosoma japonicum, Trichinella spiralis, and Neospora caninum.

BACKGROUND:The property of dihydroartemisinin (DHA) in promoting host immunohomeostasis, apart from its potent antimalarial activity, has been well-recognized. However, the mechanism of DHA in activating macrophages to enhance host resistance to malaria remains unexplored. PURPOSE:This study investigated the molecular mechanism by which DHA promotes the polarization of macrophages toward the M1 phenotype during the treatment of malaria. METHODS:The mouse macrophage cell line RAW 264.7 or the macrophages isolated from mice were stimulated with Plasmodium berghei ANKA infected red blood cells (iRBC) in the presence of DHA. The macrophage phenotypes in both in vivo and in vitro were determined using cytometric bead array and flow cytometry. To dissect the molecular mechanisms underlying macrophage responses to DHA, we initially profiled the expression of 90 genes associated with innate immunity, including the entire NLR family, in macrophages stimulated with DHA. This targeted screen strikingly revealed that only Nlrp12 was significantly upregulated among all tested NLR genes. The function of Nlrp12 was further dissected by Nlrp12 knockdown in macrophages with recombinant lentiviruses encoding Nlrp12-specific shRNA, within the context of DHA treatment. To comprehensively define the molecular consequences of Nlrp12 deficiency, we performed an integrated analysis by combining single-cell RNA sequencing with label-free quantitative proteomic profiling. This allowed us to systematically characterize the complex transcriptomic and proteomic dynamics in DHA-treated macrophages upon Nlrp12 deletion. RESULTS:DHA induced macrophage polarization to M1 phenotype and enhanced phagocytosis by up-regulating the expression of NLRP12. Nlrp12-knockdown in macrophages reduced the expression of M1 type-associated genes, resulting in a significantly increased expression of the translocator protein (TSPO), which suppressed the secretion of inflammation-associated cytokines and blunting macrophage M1 polarization. The results of single cell RNA sequencing further revealed that DHA promoted the conversion of classical M1 macrophages into lipocalin-2 (Lcn2) high M1 macrophages. CONCLUSION:The activation of NLRP12 induced by DHA is crucial for M1 macrophage polarization, which plays a significant role in the clearance of Plasmodium parasites.

Superoxide dismutases (SODs) maintain redox homeostasis through the catalytic dismutation of superoxide anions, thereby affording protection to organisms against oxidative damage. The SOD family, encompassing Cu/Zn-SOD, Mn-SOD, Fe-SOD, and Ni-SOD, exhibits structural diversity and constitutes a multilevel antioxidant defense system with discrete subcellular localizations. Beyond their antioxidant functions, SODs also function as immunomodulatory proteins, regulating the maturation, proliferation, and differentiation of immune cells. They further fulfill a crucial role in host responses to parasitic infections. The current review synthesizes and critically evaluates extant research to comprehensively delineate the molecular architecture of SODs, their intricate post-translational modification (PTM) networks, and their dual regulatory mechanisms at the interface of immunomodulation and pathological processes. This review establishes a critical framework for elucidating the biological significance of redox homeostasis maintenance.

Augmented regulatory B cell (Breg) responses are commonly observed in malaria; however, the specific parasite components and Breg subtypes involved remain unclear. In this study, we investigated C57BL/6 mice infected with Plasmodium berghei ANKA, which induces cerebral malaria pathology, in comparison to P. yoelii YM, which does not. We found that distinct Breg types differentiated in response to these infections, driven by hemozoin-mediated Toll-like receptor 9 activation. Interleukin-35-positive (IL-35+) Breg expansion occurred in P. yoelii YM-infected mice but not in those infected with P. berghei ANKA. We demonstrated that stimulator of interferon genes (STING)-mediated interferon regulatory factor 3 (IRF3) phosphorylation suppressed IL-35+ Breg differentiation, potentially contributing to experimental cerebral malaria (ECM). In contrast, P. yoelii YM infection activated IRF3 in a STING-independent manner, promoting IL-35+ Breg expansion. These findings highlight IL-35+ Bregs as key modulators in malarial immunopathology.

The invasion of Plasmodium merozoites into host erythrocytes is initiated through specific ligand–receptor interactions. This interaction results in subsequent invasion events, facilitated by the formation of a moving junction via AMA-1 and associated molecular complexes. Previous studies have implicated erythrocyte surface glycosaminoglycans, particularly heparan sulfate proteoglycans, as critical receptor components in this invasion process. The binding affinity of the PbGAC protein to heparin and erythrocytes was assessed through western blotting, immunofluorescence, flow cytometry techniques, and heparinase II treatment. Mice were immunized with the recombinant PbGAC-His to generate specific polyclonal antibodies for subcellular localization, passive immunization, and immunoprecipitation. Global mass spectrometric analyses were conducted to identify its interacting proteins. We elucidated the molecular function of PbGAC (encoded by PbANKA_1137800), a previously uncharacterized Plasmodium berghei ANKA protein, in association with merozoite attachment and invasion via the heparan sulfate-dependent pathway. The PbGAC protein, predominantly located at the extreme apical region of the P. berghei merozoite, binds to heparin and the erythrocyte surface during merozoite invasion. Global mass spectrometric analysis reveals that PbGAC interacts with several secreted proteins that are critically involved in erythrocyte invasion. In addition, mice either immunized with the PbGAC protein or passively immunized with sera derived from vaccinated mice demonstrated enhanced immunity against lethal challenges. Our findings pinpointed that PbGAC is predominantly expressed at the extreme apical region of the P. berghei merozoite and engaged in binding to the heparin-like receptors on the erythrocyte surface during merozoite invasion.

Introduction Sweetpotato (Ipomoea batatas (L.) Lam.) is a genetically intricate hexaploid crop. The purple-fleshed variety, enriched with anthocyanin pigments, is an outstanding source for creating high-value functional products. Previous research on anthocyanin biosynthesis has primarily focused on the above-ground plant parts at the transcriptional level. However, the regulatory mechanisms underlying anthocyanin accumulation in underground tuberous roots of sweetpotato remain largely unexplored. Objectives This study aimed to elucidate the post-transcriptional and post-translational mechanisms of Ib-miR2111 and its target gene IbKFB in anthocyanin synthesis in sweetpotato. Methods Genetic manipulation techniques were used to validate the function of Ib-miR2111 and IbKFB in anthocyanin biosynthesis in sweetpotato. To investigate how IbKFB works, a series of protein interaction assays, including yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), GST pull-down, co-immunoprecipitation (Co-IP), and ubiquitination, were conducted. Additionally, the impact of anthocyanin extracts from the genetically modified sweetpotato lines on inflammatory cells morphology, cytokine expression, and cell proliferation were evaluated using in vitro assays. Results Purple-fleshed sweetpotato (PFSP) varieties exhibited elevated Ib-miR2111 expression compared to white-fleshed sweetpotato (WFSP) varieties, with an inverse expression pattern in IbKFB. Genetic manipulations, including overexpression, CRISPR/Cas9 knockouts, and targeted mutations, confirmed their critical roles in anthocyanin modulation. Furthermore, IbKFB’s interactions and ubiquitination with phenylalanine ammonia-lyase 1 (IbPAL1) and glyceraldehyde-3-phosphate dehydrogenase 1 (IbGAPCp1) were elucidated, revealing intricate regulatory mechanisms. Enhanced anthocyanin content showed significant effects on inflammatory cell morphology, cytokine expression, and cell proliferation. Conclusion This study provides new insights into the regulatory mechanisms of Ib-miR2111 and IbKFB in anthocyanin biosynthesis and suggests potential health benefits of anthocyanin-rich sweetpotatoes.