Qingfeng Zhang

Transmission of malaria parasites to mosquito vectors relies on the successful conversion from asexual blood-stage forms into sexual gametocytes. Protein ubiquitination regulates many cellular processes, including cell differentiation in other eukaryotes, but whether specific ubiquitination machinery mediates Plasmodium sexual conversion is unclear. Here we conduct CRISPR–Cas9 mutagenesis screens to systematically profile Plasmodium yoelii E2 ubiquitin-conjugating enzymes for their impact on parasite development. We demonstrate that an E2 enzyme (Rad6B), and its partner RING-type E3 ligase (Tex1), play determinant roles in gametocytogenesis and transmission to mosquitoes. Deletion of either Rad6B or Tex1 results in severe defects in gametocyte production and oocyst formation. The Rad6B–Tex1 complex catalyses histone H2B monoubiquitination in Plasmodium yoelii and Plasmodium falciparum. H2B monoubiquitination promotes H3K4 tri-methylation occupancy at the promoter region of the gene encoding transcription factor AP2-G, a master regulator of sexual conversion, thereby inducing ap2-g expression and initiating sexual commitment. These findings reveal a key epigenetic mechanism leading to sexual differentiation in both human and rodent malaria parasites, providing 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.

Schistosomiasis morbidity and mortality are primarily driven by egg-triggered granulomas with subsequent fibrosis. However, the dynamics of cellular composition within these granulomas, particularly the identity and function of key immune cells that orchestrate their formation and evolution, remain poorly understood. Here, we combine single-cell RNA sequencing with multiplex immunofluorescence to characterize the hepatic immune landscape in Schistosoma japonicum -infected mice and further validate the functional roles of two neutrophil subsets. We find that eggs alter hepatic immune cell composition, which is characterized by extensive neutrophil recruitment and differentiation. Neutrophil recruitment correlates with CXCL2 derived from both an autocrine loop and paracrine signaling from monocytes. Cellular localization analysis reveals that granulomas progress through three distinct developmental stages: early Ly6G ^hi F4/80 ^lo Desmin ^lo , developing Ly6G ^mid F4/80 ^hi Desmin ^mid , and advanced Ly6G ^lo F4/80 ^mid Desmin ^hi . Neutrophil subsets display a zonal distribution within granulomas, with CD177 ⁺ neutrophils surrounding the eggs and Ltf ⁺ neutrophils localizing to the mid-outer layer. Cd177 knockdown reduces granuloma size and fibrosis, whereas Ltf suppression increases these pathological features, indicating that CD177 and LTF exert opposite effects on granuloma formation. Our findings provide new insights into the cellular complexity of S. japonicum egg-induced granulomas and may help guide the development of novel therapies for liver fibrosis.

Antimalarial drug resistance poses a critical threat to global malaria control efforts. Despite the continuous development of novel antimalarial compounds,

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.

Background: Plasmodium falciparum merozoite invasion of erythrocytes is an essential step in the asexual blood-stage cycle and a major target for antimalarial intervention. Rhoptry neck proteins play key roles in the formation and function of the tight junction, yet many remain poorly characterized. RALP1, a conserved rhoptry neck-associated leucine zipper-like protein, has been proposed to participate in erythrocyte binding and invasion. Conventional gene disruption attempts have been unsuccessful, suggesting that RALP1 may be essential for parasite survival. Nevertheless, its precise role and broader molecular impact during intraerythrocytic development remain to be fully elucidated. Methods: We generated a 3 × HA-tagged conditional knockdown line ( ralp1-ha-glmS ) using CRISPR-Cas9-mediated homologous recombination. RALP1 abundance and subcellular localization were evaluated by Western blotting and immunofluorescence assays. Effects on parasite growth, schizont maturation, merozoite invasion, and merozoite numbers were assessed using tightly synchronized cultures and established invasion and cytological assays. Transcriptomic changes following GlcN-induced RALP1 knockdown were analyzed by RNA-seq at early ring and schizont stages. Sequence-based structural and epitope features were examined using IUPred2A, ANCHOR2, AlphaFold3, NetMHCpan, and NetMHCIIpan. Results: Precise integration of the ha-glmS cassette enabled GlcN-inducible reduction of RALP1 protein levels, most prominently in schizonts. RALP1 knockdown reduced parasite proliferation, impaired schizont maturation, decreased merozoite numbers, and lowered erythrocyte invasion efficiency. RNA-seq showed limited effects in early rings but widespread downregulation of invasion- and host-parasite interaction-related genes in schizonts after correction for glucosamine-responsive transcripts, with GO enrichment highlighting processes related to host cell interaction, biological adhesion, and membrane-associated components. Sequence-based analyses indicated that RALP1 contains extensive intrinsically disordered regions with multiple predicted interaction motifs, while predicted B- and T-cell epitope hotspots concentrated within the C-terminal RBC-binding domain. AlphaFold3 modeling yielded low global confidence (pTM = 0.23), consistent with a primarily disordered architecture. Conclusions: RALP1 is required for normal schizont maturation and efficient erythrocyte invasion in P. falciparum . Its partial knockdown perturbs transcription of key invasion ligands and apical components, indicating a broader role in preparing merozoites for host-cell entry. The extensive disorder, epitope-rich C-terminal region, and essential function of RALP1 highlight its potential as a candidate for therapeutic or vaccine targeting. Graphical

Artemisinin antimalarial drugs initially exhibited remarkable efficacy against Plasmodium falciparum . However, their poor solubility and low bioavailability necessitate high doses and lead to an extremely short in vivo half-life. These limitations not only drive the emergence of drug-resistant Plasmodium strains but also compromise long-term therapeutic outcomes. Herein, we report a zein-based sustained release formulation, wherein zein, a natural maize protein, serves as a biocompatible nanocarrier to effectively encapsulate artemisinin (ART). Notably, this nanocarrier formulation achieves a 200-fold enhancement in ART’s water solubility, addressing a key bottleneck of ART-based therapies. In vitro assays confirm that the zein-based formulation allows for the sustained release of ART, which could help maintain therapeutic concentrations over extended periods and displayed different release rates and good dispersibility in both acid and basic environments. Importantly, in vitro evaluations also demonstrate that the nanoformulation exerts potent inhibitory effects against ART-resistant P. falciparum strains in both ring survival assay and recrudescence assay, attributed to the sustained maintenance of effective ART concentrations. In vivo studies, utilizing both rodent malaria models and humanized erythrocyte mouse models, further validate the nanoformulation’s therapeutic potential. The zein nanocarrier significantly prolongs ART’s in vivo half-life via its sustained-release capability, thereby maintaining effective blood concentrations over an extended duration. Compared to free ART, the nanoformulation exhibits superior efficacy in reducing parasitemia, preventing malaria recrudescence, and, most notably, overcoming ART resistance in drug-resistant Plasmodium infections. Collectively, these findings establish the zein-based nanocarrier as a promising strategy to optimize ART-based therapies by addressing solubility and pharmacokinetic limitations while effectively combating drug-resistant malaria. IMPORTANCE: Half of the world’s population is at risk of malaria infection, and artemisinin (ART) turns out to be a powerful medicine for malaria control. The rapid emergence and global spread of resistance to ART have led to a significantly increasing clinical treatment failure rate worldwide. A critical limitation of ART is its extremely short blood half-life (~1 h), which results in rapid declines in plasma drug concentrations below therapeutic thresholds. Some parasites may switch into a “dormant” form, which is less sensitive to ART, resulting in recrudescence following treatment. Thus, developing a sustained-release formulation provides a promising solution to prolong the in vivo half-life of ART. Additionally, its relatively low solubility restricts its in vivo bioavailability, primarily due to the limited dissolution and absorption of the compound in aqueous biological environments. In this study, we prepared a zein-based sustained-release formulation of ART for oral and intraperitoneal administration. Our results indicate that this zein-based sustained release nanoformulation not only significantly improves ART’s water solubility (a key barrier to its bioavailability) but also extends its in vivo half-life via controlled drug release. Importantly, the prolonged half-life ensures sustained therapeutic ART concentrations, directly enhancing the formulation’s ability against ART-resistant P. falciparum strains. Collectively, these results highlight the formulation’s substantial potential for clinical application in improving ART-based antimalarial therapy.

Schistosomiasis morbidity and mortality are primarily driven by hepatic granulomas triggered by parasite eggs with subsequent periportal fibrosis. As a common pathological consequence of various chronic liver injuries, hepatic fibrosis is well-documented. However, the dynamics of cellular composition within the schistosome egg-induced granuloma, particularly the identity and function of key immune cell subsets that orchestrate its formation and evolution, remain poorly resolved. To address this, we combined single-cell RNA sequencing with multiplex immunofluorescence, histopathological analysis, and serological profiling to comprehensively characterize the hepatic immune landscape in a murine model of Schistosoma japonicum infection, and further validated the functional roles of two neutrophil subsets through targeted marker gene knockdown. We found that egg deposition altered hepatic immune cell composition, characterized by extensive neutrophil recruitment and differentiation. Neutrophil recruitment correlated with CXCL2 derived from both an autocrine loop and paracrine signaling by monocytes. Cellular localization analysis of the developmental trajectory of granulomas showed that they progressed from the early Ly6G ^hi F4/80 ^lo Desmin ^lo stage, through the developing Ly6G ^mid F4/80 ^hi Desmin ^mid stage, to the advanced Ly6G ^lo F4/80 ^mid Desmin ^hi stage. In addition, neutrophil subsets display zonal distribution within early granulomas: CD177 ⁺ neutrophils surrounded the eggs, while Ltf ⁺ neutrophils localized to the mid-outer layer. Cd177 knockdown reduced granuloma size and fibrosis, while Ltf suppression increased these pathological features, indicating that CD177 ⁺ and LTF ⁺ neutrophils have opposite effects on granuloma formation. Our findings provide new insight into the cellular complexity of S. japonicum egg-induced granulomas and could help guide the development of novel treatments for liver fibrosis.

Obligate intracellular parasites must efficiently invade host cells to complete their life cycle and facilitate transmission. For the malaria-causing parasite Plasmodium falciparum, the invasion of an erythrocyte is a critical process, and thereby a key target for intervention strategies. In this study, we investigate the role of the ApiAP2 family transcription factor PfAP2-06B (PF3D7_0613800) in the intraerythrocytic developmental cycle of P. falciparum and focus on its regulation of genes involved in erythrocyte invasion. Conditional knockdown of PfAP2-06B resulted in a defect in asexual growth and impaired erythrocyte invasion. Bulk RNA sequencing (RNA-seq) analysis revealed that PfAP2-06B modulates the expression of invasion-related genes during the schizont stage. Single-cell RNA sequencing indicated that PfAP2-06B influences invasion gene expression and contributes to stochastic variations in expression of cell-to-cell genes. These results underscore the critical function of PfAP2-06B in the process of erythrocyte invasion and suggest its potential as a target for novel malaria control strategies. Importance: Understanding gene regulation in Plasmodium falciparum is essential for uncovering mechanisms of parasite development and pathogenicity. The research underscores the pivotal role of PfAP2-06B in regulating critical aspects of Plasmodium intraerythrocytic development and host cell invasion, demonstrating that PfAP2-06B plays a key role in orchestrating stage-specific gene expression. These findings provide new insights into the transcriptional networks of P. falciparum and highlight PfAP2-06B as a potential target for therapeutic intervention. This work advances our understanding of malaria pathogenesis and developing effective interventions.

Artemisinin and its semisynthetic derivatives (ART) are crucial medicines in artemisinin-based combination therapies worldwide. Despite ART’s efficacy, small proportions of young intraerythrocytic ring stage parasites can survive the drug’s short half-life, and dormant forms can cause recrudescence if not cleared by partner drugs. Certain mutations in the Kelch propeller region of P. falciparum protein (PfK13) are linked to the higher ring-stage survival (RS), which above 1% can be a feature of ‘artemisinin partial resistance’. Emerging evidence indicates epigenetic modulators may contribute to RS. Here, we report systematic evaluations of all putative histone acetyltransferases (HATs) of P. falciparum in 30 culture-adapted field isolates and 43 subcloned field isolates. Only PfMYST shows a full association with RS phenotype modulations. Knockdown experiments confirm the linkage of Pfmyst expression to these modulations, with evidence of altered metabolic processes. Through single-cell RNA sequencing, ChIP-seq analysis, and CRISPR/cas9 genetic manipulation, PfMYST-targeted RS-related genes have been identified and functionally validated. Multi-omics analysis indicates significant interplay of PfMYST and PfK13 mechanisms in RS. PfMYST epigenetic modulation extends to other antimalarials, including amodiaquine, pyrimethamine, chloroquine, and pyronaridine. Collectively, our findings provide important information on the epigenetic regulatory mechanism of P. falciparum RS after pulses of ART and other antimalarials.