https://orcid.org/0000-0003-3541-488X
Figures
Abstract
Schistosomiasis remains a critical global health issue, necessitating novel therapies due to emerging praziquantel resistance. We previously developed a patented praziquantel derivative, DW-3–15, which demonstrated potent broad-spectrum schistosomicidal activity through SjHAT1 inhibition. Here, the full-length SjHAT1 cDNA is cloned by rapid amplification of cDNA ends methods. The protein encoded by the cDNA retains conserved catalytic residues of acetyltransferases although sharing 34% identity with mammalian orthologs. Enzyme activity analysis reveals that recombinant SjHAT1 exhibits 130 μU/mL histone acetyltransferase activity at 25°C for 60 min, and is completely inhibited by DW-3–15 at a concentration of 50 μM. However, DW-3–15 has no effect on the enzymatic activity of the human ortholog HAT1. Phylogenetic analyses place SjHAT1 in a distinct clade, and molecular docking results indicate significant divergence compared to its human ortholog HsHAT1. The expression profiling of SjHAT1 reveals stage- and sex-specific patterns. Fluorescence in situ hybridization localizes SjHAT1 predominantly in female vitellaria and male parenchyma near the gynecophoral canal. Knockdown of SjHAT1 reduces worm survival by 57.5% in vivo, suppresses female oviposition by 90.8% in vitro and 79.2% in vivo, and disrupts ovarian and vitelline architecture. RNA-sequencing analysis reveals that SjHAT1 knockdown disrupts β-alanyl-tryptamine (BATT) pheromone signaling via downregulation of aromatic L-amino acid decarboxylase (AADC) in males and multidrug resistance-associated protein 4 (MRP4) in females. Given that BATT is essential for inducing female sexual development and egg production, the observed transcriptional dysregulation provides mechanistic evidence for disrupted intersexual communication. Our findings identify that SjHAT1 is a central regulator of schistosome reproductive biology. These sex-specific mechanisms highlight SjHAT1 as a potential therapeutic target for treatment and control of schistosomiasis.
Author summary
Schistosomiasis is a neglected tropical disease caused by Schistosoma species. Reliance on the single drug praziquantel (PZQ) for treatment and control of schistosomiasis is not sustainable. Emerging drug resistance highlights the critical need for alternative treatments. In our previous studies, we developed a novel derivative of PZQ, designated DW-3–15, which exhibited broad-spectrum schistosomicidal activity against all developmental stages of S. japonicum in vivo, with notably potent efficacy against juveniles. Further, we observed synergistic effects when combining PZQ with DW-3–15 in vitro and in vivo, suggesting that there were distinct molecular targets for the two compounds. Later, we found that the therapeutic effect of DW-3–15 was mediated through downregulation of Schistosoma japonicum histone acetyltransferase 1 (SjHAT1). Understanding the biological function played by SjHAT1 is of great importance for developing novel anti-schistosomal drug targets. In this study, we find that SjHAT1 is predominantly expressed in the vitellaria of female worms and in the parenchyma near the gynecophoral canal of male worms. The localization of SjHAT1 in schistosomes indicates its significant role in the reproductive development and egg-laying processes of female worms. Eggs are the key pathogenic factors and are responsible for the transmission of schistosomiasis. Knockdown of SjHAT1 significantly compromises worm survival and suppresses female oviposition. Transcriptomic analysis further reveals that SjHAT1 silencing disrupts schistosome β-alanyl-tryptamine (BATT) pheromone signaling via two synergistic pathways. Specifically, male-specific suppression of aromatic L-amino acid decarboxylase (AADC) impairs BATT pheromone synthesis, thereby hindering female sexual maturity. Concurrently, female-specific downregulation of multidrug resistance-associated protein 4 (MRP4) inhibits BATT signal transduction, synergistically blocking oviposition. As BATT is essential for inducing female sexual development and egg production, these findings establish SjHAT1 as a central regulator of schistosome reproductive biology. We have validated SjHAT1 as a promising druggable target through in vitro and in vivo investigations. Further studies are required to establish SjHAT1 as a specific epigenetic regulator governing schistosome reproduction. The dual-sex regulatory mechanism provides a framework for understanding the fascinating sexual biology of schistosomes, which could lead to novel strategies for treatment and control of schistosomiasis.
Citation: Xu J, Wang Y-X, Huang P, Zhang Y-N, Sun H, Zhan T-Z, et al. (2026) Schistosoma japonicum histone acetyltransferase 1 (SjHAT1): A novel anti-schistosomal drug target. PLoS Pathog 22(6): e1014334. https://doi.org/10.1371/journal.ppat.1014334
Editor: Mostafa Zamanian, University of Wisconsin-Madison, UNITED STATES OF AMERICA
Received: December 22, 2025; Accepted: June 2, 2026; Published: June 24, 2026
Copyright: © 2026 Xu 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.
Data Availability: All data are available in the main text or the supplementary materials. The accession number of full-length SjHAT1 cDNA in NCBI database is PQ778043. The raw RNA sequencing data of male and female S. japonicum worms treated with GFP-dsRNA or SjHAT1-dsRNA have been deposited in the Sequence Read Archive (SRA) database under the accession number PRJNA1313613.
Funding: This work was supported by National Natural Science Foundation of China (No. 81902083 to J.X, No. 82172294 to C.M.X), Priority Academic Program Development of Jiangsu Higher Education Institutions (No. YX13400214 to J.X), and Key Laboratory of Basic Research on Regional Diseases (Guangxi Medical University), Education Department of Guangxi Zhuang Autonomous Region (No. GXQYJB2024001 to J.X). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Schistosomiasis is a neglected parasitic disease caused by infection with trematodes of the genus Schistosoma, mainly Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Schistosoma intercalatum, Schistosoma guineensis, and Schistosoma mekongi. Current global data indicate that approximately 1 billion people are at risk of schistosomiasis, with more than 250 million people infected across 78 countries [1,2]. Annually, about 250,000 deaths are attributed to this disease [3]. In the absence of an effective vaccine, praziquantel (PZQ) is the only available drug to control this debilitating disease [4]. However, PZQ is ineffective against immature worms and does not prevent reinfection, and repeated use of drug monotherapy in many endemic regions has raised serious concerns about the emergence of drug resistance [5,6]. Therefore, developing alternative treatments and identifying novel drug targets for schistosomiasis remains crucial.
Currently, three strategies dominate the search for new anti-schistosomal drugs: (1) synthesis and evaluation of PZQ analogues, (2) design of new pharmacophores and (3) high-throughput screening of new active compounds [7]. While designing novel pharmacophores is challenging due to the still unclear mechanism of PZQ [8], the development of PZQ derivatives remains a primary approach for discovering alternative therapeutics. In prior work, our group hybridized the endoperoxide bridge pharmacophore of artesunate (which targets juvenile schistosomes) to PZQ (which targets adults) by inserting a hydroxyl group at the position C10 of the pharmacophore aromatic ring in PZQ, yielding a novel praziquantel derivative DW-3–15 (Patent No. ZL201110142538.2) [9]. DW-3–15 exhibited broad-spectrum schistosomicidal activity against all developmental stages of S. japonicum in vivo, with especially high efficacy against juveniles [10]. The anti-juvenile and anti-adult effects of DW-3–15 were consistently observed in commercially synthesized batches, confirming its structural stability. Moreover, DW-3–15 significantly reduced egg deposition by female worms and mitigated hepatic pathology, further supporting its therapeutic potential [11]. Notably, combining PZQ with DW-3–15 produced synergistic effects in vitro and in vivo; even at sub-therapeutic doses, the combination achieved efficacy comparable to or exceeding either monotherapy, suggesting the two compounds have distinct molecular targets [12].
To investigate the molecular mechanism of DW-3–15, we performed tandem mass tag (TMT) quantitative proteomics on treated worms. This analysis revealed that S. japonicum histone acetyltransferase 1 (SjHAT1) was one of the most significantly downregulated proteins in female worms following in vivo DW-3–15 treatment [13]. HAT1 is a member of the GCN5-related N-acetyltransferases (GNAT) superfamily and is one of the first histone acetyltransferases discovered, primarily acetylating lysine 5 and lysine 12 of histone H4 (H4K5 and H4K12) [14]. HATs catalyze the transfer of acetyl groups to lysine residues on histones, thereby increasing chromatin accessibility to promote gene transcription [15]. This acetylation-mediated post-translational modification plays crucial roles in the growth, development, and reproduction of schistosomes [16–19]. However, only few investigations on HAT inhibitors against schistosomes are currently available. Carneiro et al [18] demonstrated that histone acetylation by SmCBP1 and SmGCN5 in S. mansoni establishes an epigenetic state necessary for Smp14 gene activation and eggshell formation; the HAT inhibitor PU139 prevents chromatin decondensation at the Smp14 promoter, thereby impairing egg production and disrupting female reproductive development [18]. Furthermore, testing of the HAT inhibitors A485, C646, and curcumin identified curcumin as a potent schistosomicidal agent that dose-dependently suppresses SjHAT1 expression. Notably, we also found that DW-3–15 could inhibit SjHAT1 expression, implicating SjHAT1 as a promising target mediating its anti-schistosomal effects [13].
Despite these findings, the biological roles of SjHAT1 in S. japonicum, particularly its involvement in drug-mediated killing of worms and in regulating female reproductive activity, remain poorly characterized. In this study, we employed rapid amplification of cDNA ends (RACE) to systematically analyze the bioinformatics features of SjHAT1. Subsequently, we investigated its functions both in vitro and in vivo. Finally, we utilized RNA sequencing (RNA-seq) to explore the regulatory role of SjHAT1 in downstream signaling pathways, thereby establishing experimental and theoretical foundations for the development of SjHAT1-targeted anti-schistosomal interventions.
Results
SjHAT1 is a promising drug target against schistosomiasis
Since the cDNA sequence of SjHAT1 in the NCBI database is predicted and incomplete, we utilized the rapid amplification of cDNA ends (RACE) technique to obtain its full-length cDNA sequence for further characterization. The full-length cDNA sequence of SjHAT1 was successfully obtained through RACE assay, revealing a 1302 bp open reading frame (ORF) encoding 433 amino acids (NCBI accession no. PQ778043). Using the InterPro database, we identified that SjHAT1 contains an N-terminal HAT1 domain (IPR019467, 14–175aa) and a C-terminal HAT type B catalytic subunit domain (IPR048776, 316–339aa). The N-terminal HAT1 domain catalyzes the transfer of an acetyl group from acetyl-CoA to lysine residues at the histone N-terminus [20]. By contrast, the C-terminal domain is not conserved and is dispensable for HAT1 catalytic activity [21]. To investigate the enzymatic function of SjHAT1, we successfully expressed recombinant SjHAT1 (rSjHAT1) in E. coli and demonstrated its robust histone acetyltransferase activity through in vitro assays (Table 1). Notably, the enzymatic activity of rSjHAT1 was dose-dependently inhibited by the compound DW-3–15, with complete inhibition at a concentration of 50 μM (Table 1). Meanwhile, DW-3–15 had no effect on the enzymatic activity of Homo sapiens rHAT1 (S1 Table). To validate whether SjHAT1 has potential as a drug target, we performed a multiple sequence alignment of SjHAT1 with HAT1 homologs from Homo sapiens, Mus musculus, Rattus norvegicus, Schistosoma mansoni, Schistosoma haematobium, Clonorchis sinensis, and Fasciola hepatica. Multi-sequence alignment revealed that SjHAT1 shares approximately 34% sequence identity with the HAT1 proteins of human, mouse, and rat, and the key amino acids required for HAT1 enzymatic activity are highly conserved (Fig 1A). Phylogenetic analysis demonstrates that SjHAT1 clusters in an evolutionarily distinct clade, and displays significantly higher sequence homology with HAT1 orthologs from other trematode species compared to non-parasitic flatworms, underscoring strong evolutionary conservation within parasitic flatworms (Fig 1B). Structural characterization of motif architecture further indicates that critical functional domains exhibit remarkable conservation across taxa (Fig 1B). Evolutionarily conserved protein core domains maintain functional integrity, while different spatial conformations facilitate functional specialization. Therefore, we employ AlphaFold3 to predict the tertiary structure of SjHAT1 (S1 Fig and S2 Table) and conduct a comparative analysis with its human homolog HsHAT1 (PDB: 2P0W). Structural superposition reveals that SjHAT1 and HsHAT1 share a conserved fold architecture, as evidenced by their significant global structural similarity (TM-score = 0.92), while exhibiting notable local divergence (RMSD = 2.40 Å). Molecular docking results reveal that among the top 100 docking conformations, the SjHAT1-DW-3–15 complex demonstrates heterogeneous binding modes, whereas the HsHAT1-DW-3–15 complex exhibits limited conformational diversity (Fig 1C). Furthermore, residue-level interaction analysis identifies distinct binding patterns. In the optimal binding model, we selected amino acids within 4 Å of the DW-3–15. SjHAT1 engages the ligand primarily through Trp-187, Tyr-215, Phe-217, Tyr-218, Tyr-220, Arg-227, and Glu-266 (Fig 1D), whereas HsHAT1 employs Gly-230, Gln-231, Ala-235, Glu-239, Lys-265, Leu-266, Asp-268, Phe-269, and Ile-315 (Fig 1E). Electrostatic potential mapping shows a significantly more electronegative binding cavity in SjHAT1 compared to HsHAT1. This charge disparity likely mediates divergent ligand-binding thermodynamics through modulation of ligand orientation and binding free energy (Fig 1F). These distinctive molecular characteristics, particularly the structural divergence from HsHAT1, establish SjHAT1 as a promising druggable target for schistosomiasis. Such divergence suggests that species-specific targeting of SjHAT1 could mitigate host toxicity by avoiding interference with the host’s HAT1.
(A) Multiple sequences alignment of SjHAT1. Red box indicates the N-terminal domain of HAT1, blue box indicates the C-terminal domain of HAT1, and yellow triangles highlight key amino acids experimentally validated to be associated with HAT1 protein activity in other species (B) phylogenetic analysis of SjHAT1. SjHAT1 is shown in red. The right panel illustrates the structural organization and phylogenetic distribution of conserved motifs within HAT1 orthologs across different species. (C) Computational characterization of SjHAT1/HsHAT1and DW-3-15 interactions using molecular docking and ensemble analysis. Structural ensemble analysis was performed on the top 100 docking poses ranked by interface-predicted TM (ipTM) score through a computational modeling framework. Ligand conformations were chromatically coded according to binding energy gradients, while target proteins SjHAT1/HsHAT1 were displayed as monochromatic surface representations with transparency-adjusted secondary structures. (D) Three-dimensional structural model of SjHAT1 with the highest ipTM score and its ligand-binding site. The DW-3-15 ligand represented as magenta sticks. Close-up view of the ligand-binding pocket, highlighting critical interactions with key amino acid residues (Trp-187, Tyr-215, Phe-217, Tyr-218, Tyr-220, Arg-227 and Glu-266). (E) Three-dimensional structural model of HsHAT1 with the highest ipTM score and its ligand-binding site. The DW-3-15 ligand represented as pink sticks. Close-up view of the ligand-binding pocket, highlighting critical interactions with key amino acid residues (Gly-230, Gln-231, Ala-235, Glu-239, Lys-265, Leu-266, Asp-268, Phe-269, Ile-315). (F) Comparison of surface electrostatic potential distributions between SjHAT1 and HsHAT1 Proteins. Red areas denote negative electrostatic potential regions, blue areas correspond to positive electrostatic potential regions, and white areas represent neutral zones. The scale bar beneath indicates the corresponding electrostatic potential range (kT*e-1).
SjHAT1 is highly expressed in the vitellaria of adult female worms
To further characterize SjHAT1 and facilitate future investigations into its physiological functions, the mRNA expression profiles of the SjHAT1 across six critical developmental stages, including eggs, cercariae, 14-day-old juveniles, 21-, 28- and 35-day-old adult male/female worms were quantified. The results revealed that SjHAT1 transcripts, while ubiquitously expressed, exhibited significant stage specificity and sexual dimorphism. The highest expression level of SjHAT1 was observed in 35-day-old females, whereas the lowest expression was detected in eggs. Notably, sex-specific differences became pronounced in 28- and 35-day-old adults, with SjHAT1 expression in females significantly exceeding that in males (p < 0.0001, Fig 2A). This differential expression pattern suggests that SjHAT1 may play a pivotal role in sexual maturation and female reproductive development in schistosomes.
(A) Transcriptional levels of SjHAT1 in different developmental stages and genders of S. japonicum. N = 3 biological replicates, Student’s t-test, standard deviation (SD) is shown in the error bars. ‘ns’ indicates no significant difference (p > 0.05), ****p < 0.0001. The raw data is shown in supporting information file [Table B in S1 Data]. (B) Localization of SjHAT1 in female S. japonicum. ‘ov’, ovary; ‘vg’, vitelline gland. Scale bars: 200 μm. (C) Localization of SjHAT1 in male S. japonicum. ‘t’, testis; ‘p’, parenchymal cell. Scale bars: 200 μm.
Subsequently, we investigated the tissue-specific localization of SjHAT1 in 35-day-old adult worms. Fluorescence in situ hybridization (FISH) revealed that SjHAT1 is predominantly expressed in the vitellaria of female worms (Fig 2B) and in the parenchymal cells adjacent to the gynecophoral canal of male worms (Fig 2C). The female-biased expression pattern in vitellaria, a specialized organ responsible for vitellocytes production [22], further implicates SjHAT1 in reproductive regulation.
SjHAT1 knockdown impairs viability and oviposition of adult S. japonicum in vitro
To investigate the functions of SjHAT1, we conducted RNA interference (RNAi) in paired adult worms in vitro. We designed two dsRNAs, SjHAT1-dsRNA1 and SjHAT1-dsRNA2, targeting the N-terminal and C-terminal domains of SjHAT1, respectively. Through preliminary experiments comparing the interference efficiency of SjHAT1-dsRNA1 (30 μg/mL), SjHAT1-dsRNA2 (30 μg/mL), and SjHAT1-dsRNA1/2 (15 μg/mL SjHAT1-dsRNA1 and 15 μg/mL SjHAT1-dsRNA2), we found that the SjHAT1-dsRNA1/2 exhibited the highest interference efficiency (S2 Fig). Hence, in the following studies, SjHAT1-dsRNA1/2 was added on day 1, day 3, and day 7 using the soaking method [3]. The results showed that in vitro RNAi significantly reduced both the transcriptional level and protein expression of the SjHAT1 in female and male worms (Fig 3A and 3B).
(A) Changes in SjHAT1 transcript level after SjHAT1-dsRNA treatment. Standard deviation (SD) is shown in the error bars, n = 3 bilogical replicates, Student’s t-test, ***p < 0.001, ****p < 0.0001. (B) Changes in SjHAT1 protein expression after SjHAT1-dsRNA treatment. Standard deviation (SD) is shown in the error bars, n = 3 biological replicates, **p < 0.01. (C) Altered oviposition in females after SjHAT1-dsRNA treatment. Error bars demonstrate standard deviation (SD), n = 12, Two-Way ANOVA, *p < 0.05, ****p < 0.0001. The raw data is shown in supporting information file [Table C in S1 Data]. (D) Altered Motility in females after SjHAT1-dsRNA treatment. Error bars represent standard deviation (SD), n = 40, Two-Way ANOVA, *p < 0.05, ****p < 0.0001. (E) Altered Motility in males after SjHAT1-dsRNA treatment (n = 40), Two-Way ANOVA, *p < 0.05, ****p < 0.0001. The raw data is shown in supporting information file [Table D in S1 Data]. (F) Morphological changes in the reproductive organs of worms after SjHAT1-dsRNA treatment. ‘t’, testis; ‘ov’, ovary; ‘vg’, vitelline gland. Scale bars: 20 μm.
After 7 days of SjHAT1-dsRNA treatment, S. japonicum adult worms exhibited pronounced phenotypic alterations, including markedly reduced egg laying by females and compromised locomotor activity. As shown in Fig 3C, female oviposition was significantly reduced at day 5 posttreatment with SjHAT1-dsRNA interference (p < 0.05), reaching the lowest at day 7 (p < 0.0001). The reduction rate of female oviposition was 90.8% after 7 days interference. Similarly, both female and male worms exhibited significantly lower viability compared with the control group from day 3 onward (Fig 3D and 3E, p < 0.05), with scores reaching their lowest levels by 7 days posttreatment (Fig 3D and 3E, p < 0.0001). At day 7, the intervention showed no statistically significant impact on worm pairing (S3 Fig).
To assess reproductive organ development following SjHAT1 knockdown, worms were carmine-stained and examined by confocal laser scanning microscopy (CLSM). We found that knockdown of SjHAT1 severely disrupted the morphology of the reproductive organs in both sexes of S. japonicum. In males, the testes became disorganized, with indistinct lobular boundaries, loose stromal matrices, reduced spermatogonial populations, and abnormal cellular morphology. In females, the vitelline glands were atrophic, characterized by effaced lobular boundaries, fewer mature vitelline cells, and markedly shrunken ovaries (Fig 3F).
It is well known that an intact tegument is crucial for worm survival [7]. Disruption of the tegument leads to exposure of the worm’s antigens, rendering it more susceptible to the host immune system [23,24]. We therefore employed scanning electron microscopy (SEM) to examine the tegumental changes after SjHAT1 knockdown. According to the in vitro experimental results, worm viability began to decline significantly by 3 days post-interference. Consequently, parasites collected on the third day after interference were subjected to SEM observation. Under SEM, the tegument of the female worm in the control group remained intact, with spines uniformly arranged in the ventral sucker (S4 Fig). In contrast, following SjHAT1-dsRNA intervention for 3 days, distinct tegumental damage was evident. The anterior region of female worms exhibited mild swelling accompanied by numerous blisters (Fig 4B). The mid-body tegument displayed extensive sloughing (Fig 4C), and obvious fusion of the spines occurred in the ventral suck of females (Fig 4D). Silencing SjHAT1 induces morphological changes (Fig 4) similar to those observed after 72 h treatment with 100 μM DW-3–15, although the severity of damage is less pronounced comparable to the DW-3–15 treatment group (S5 Fig). These in vitro results indicate that SjHAT1 knockdown critically impairs survival and reproductive functions in adult S. japonicum.
(A) The whole worm is swollen and shrunken; (B) mild swelling accompanied by numerous blisters is observed at the anterior end of female worm; (C) extensive sloughing is observed on the tegument in the mid-body of female worm; (D) fusion of the spines in the ventral sucker are obvious; (E)-(F) no significant changes are observed in the transverse ridge-like structures on the tegument of female worm. The ridges arranged orderly accompanied by uniformly distributed pores. Scalebars: A: 1 mm; B: 100 μm; C: 100 μm; D: 5 μm; E: 5 μm; F: 3 μm.
SjHAT1 is essential for the survival, pairing and oviposition of S. japonicum in vivo
Our in vitro results indicate SjHAT1 is required for maintaining the adult worm viability and is crucial for reproductive processes in S. japonicum. However, the in vitro SjHAT1-dsRNA interference experiment was conducted on paired adult worms and did not address the role of SjHAT1 at other developmental stages. Furthermore, it is technically challenging to collect and culture schistosomula of different developmental stages in vitro. Thus, we carried out in vivo RNAi experiments to assess the effects of SjHAT1 knockdown across parasite developmental stages. Mice infected with S. japonicum cercariae received a tail vein injection of 10 μg SjHAT1-dsRNA at 14 days post-infection (dpi), followed by repeated injections of 10 μg dsRNA at 18, 22, 26, and 30 dpi. On day 35 post-infection, the parasites were harvest through perfusion of the hepatic portal system and mesenteric veins, and mouse livers were collected for egg counting and histopathological analysis (H&E staining). Worm burden reduction and hepatic egg burden reduction were calculated.
In these experiments, mice that received control GFP-dsRNA showed no significant differences in worm or egg burdens compared to blank controls injected with saline (S6A, S6B and S6E Fig). Parasites recovered from GFP-dsRNA control mice were morphologically normal (S6C and S6D Fig), and there were no significant differences in the number or size of liver egg granulomas between GFP-dsRNA and blank control groups (S6F and S6G Fig). These findings confirm that GFP-dsRNA had no appreciable effect on the parasites. In contrast, mice treated with SjHAT1-dsRNA showed a 57.5% reduction in worm burden (Fig 5A, p < 0.0001) and a 65.9% reduction in pairing (Fig 5B, p < 0.0001) compared to the GFP-dsRNA controls. Moreover, many female worms from the SjHAT1 knockdown group exhibited developmental arrest (Fig 5C), with 32.9% remaining immature, which was significantly higher than that the 18.9% observed in the GFP control group [χ2(1) =5.528, p = 0.0187, S3 Table]. These results indicate that the downregulation of SjHAT1 impairs the development and sexual maturity of female worms, thereby reducing their egg-laying capacity. Consistent with these observations, CLSM analysis revealed severe testicular hypoplasia in males, while females showed ovarian degeneration and vitelline gland atrophy characterized by diminished lobular boundaries and reduced mature vitellocyte count (Fig 5D). Eggs are the key factor responsible for the pathogenesis of schistosomiasis [25–28]. The formation of hepatic egg granulomas depends not only on the number of deposited eggs but also on their viabilities. Pathologically, SjHAT1-RNAi livers displayed significantly attenuated hepatic granuloma formation (Fig 5E) and 79.2% reduction in egg deposition (p < 0.001, Fig 5F). H&E staining confirmed 82.9% smaller granuloma areas 9480 ± 5029 μm2 vs control 55542 ± 17398 μm2 (p < 0.001, Fig 5G and 5H), demonstrating impaired oviposition capacity post-RNAi. These findings indicate that SjHAT1 knockdown in vivo severely impairs oviposition and significantly mitigates egg-induced hepatic pathology.
On the 14th, 18th, 22th, 26th and 30th days postinfection, SjHAT1-dsRNA was injected through the tail vein, and the hepatic portal vein was perfused on the 35th day. (A) Worm burden of the parasites recovered at 35 dpi in GFP control RNAi and SjHAT1 RNAi groups. N = 3 biological replicates, 3 mice of each biological replicates. Student’s t-test, ****p < 0.0001. The raw data is shown in supporting information file [Table E in S1 Data]. (B) Comparison of the pairing rates of recovered worms between GFP and SjHAT1 dsRNA groups. N = 3 biological replicate, 3 mice of each biological replicates. Student’s t-test, ****p < 0.0001. The raw data is shown in supporting information file [Table F in S1 Data]. (C) Morphological observation of worms after RNAi in vivo. Scale bars: 2000 μm. (D) Morphological changes in the reproductive organs of worms after RNAi in vivo. ‘t’, testis; ‘ov’, ovary; ‘vg’, vitelline gland. Scale bars: 20 μm. (E) Gross observations of the mouse liver in the GFP and SjHAT1 dsRNA groups. Scale bars: 1 cm. (F) Eggs per gram of the liver comparation between the GFP and SjHAT1 dsRNA groups (n = 90). Student’s t-test, ****p < 0.0001. The raw data is shown in supporting information file [Table G in S1 Data]. (G) Histological assessment of mouse liver by H&E staining. Scale bars: 100 μm. (H) Statistical analysis of the size of egg granuloma area after RNAi in vivo (n = 30). Student’s t-test, ****p < 0.001. Error bars represent standard deviation (SD). The raw data is shown in supporting information file [Table H in S1 Data].
SjHAT1 knockdown modulates ABC transporter pathway in females and tyrosine metabolism in males
Having demonstrated that SjHAT1 knockdown disrupts the normal morphology, growth, development and oviposition of S. japonicum, we next investigated the molecular pathways regulated by SjHAT1. We performed RNA sequencing (RNA-seq) analysis on male and female worms following SjHAT1 knockdown in vitro. The RNA-seq results revealed a total of 206 differentially expressed genes (DEGs) in female worms (86 upregulated and 120 downregulated; Fig 6A) and 786 DEGs in male worms (242 upregulated and 584 downregulated; Fig 6B). Gene Ontology (GO) analysis and KEGG pathway enrichment were then applied to these DEGs.
(A) Volcano plot illustrating differentially expressed genes (DEGs) in female parasites. (B) Volcano plot illustrating DEGs in male worms. DEGs were defined as |log2 Fold Change| ≥ 0.58 and p-value< 0.05. (C) Heatmap of the differentially expressed genes. (D) KEGG pathway enrichment analysis of DEGs in female worms. (E) KEGG pathway enrichment analysis of DEGs in male worms. (F) Differentially expressed genes following SjHAT1 knockdown. (G) qRT-PCR validation of MRP4 (female) and AADC (male) expression. N = 3 bilogical replicates, Student’s t-test, ***p < 0.001. Error bars represent standard deviation (SD).
After the knockdown of SjHAT1, genes with the most significant expression changes included copine-9 (KSF78_0001940), calcium activated potassium channel subunit (KSF78_0002097), tyrosine-protein kinase FRK (KSF78_0003289), ribonuclease T2 (KSF78_0002281), multidrug resistance-associated protein 4 (MRP4, KSF78_0000799) and multidrug resistance protein 1 (KSF78_0005913) in females, and neuronal acetylcholine receptor subunit alpha-2 (KSF78_0006978), aromatic L-amino acid decarboxylase (AADC, KSF78_0006508), tyrosinase (KSF78_0005796, KSF78_0005182), tyrosine hydroxylase (KSF78_0002665) in males (Fig 6C and 6F).
In female worms, the downregulated DEGs were significantly enriched in the ATP binding cassette (ABC) transporter pathway (Fig 6D), with MRP4 being specifically and significantly downregulated (p = 0.01931655). Consistently, GO analysis linked this female DEG to transmembrane transport processes (GO:0055085). In male worms, the downregulated DEGs were predominantly enriched in the tyrosine metabolism pathway (Fig 6E). Notably, AADC, a gene normally highly expressed in males, was dramatically downregulated (p = 5.49458E-07). GO analysis indicated that this enzyme is involved in carboxylic acid metabolic processes (GO:0019752). Finally, qRT-PCR validation confirmed that SjHAT1 knockdown significantly reduced MRP4 expression in female worms and AADC transcriptional levels in male worms (Fig 6G), consistent with the RNA-seq data.
Discussion
In our previous work, we observed that the praziquantel derivative DW-3–15, has exceptional schistosomicidal activity against all developmental stages of S. japonicum, with >60% worm reduction against juveniles [10]. This broad efficacy contrasts sharply with PZQ, which mainly targets adult worms [29]. Notably, DW-3–15 was found to markedly suppress the expression of SjHAT1 in adult worms. Similarly, curcumin, a histone acetyltransferase inhibitor that also suppresses SjHAT1 expression, elicited phenotypic changes resembling those caused by DW-3–15 [13]. These findings suggest that SjHAT1 is a critical molecular target underlying the anti-schistosomal effects of DW-3–15. However, the existing SjHAT1 cDNA sequence in the NCBI database is incomplete and based on predictions. We used RACE to clone the full-length SjHAT1 cDNA (NCBI accession no. PQ778043) and characterized the encoded protein. We identified four nucleotide differences from the predicted sequence, including a nonsynonymous mutation (cysteine to serine) at residue 311. Phylogenetic analysis reveals that SjHAT1 forms a distinct clade separate from humans and other mammals, whereas it exhibits remarkable evolutionary conservation across trematode species (Fig 1B). Molecular docking analysis demonstrates that both the critical residues mediating the SjHAT1-DW-3–15 interaction and the surface electrostatic potential distribution showed significant divergence from corresponding features in HsHAT1 (Fig 1C–1F). This divergency implies that pharmacological agents could be designed to specifically target SjHAT1’s unique domains, potentially minimizing off-target effects on homologous host proteins and thereby mitigating toxicity. Importantly, our qPCR analysis confirmed constitutive expression of SjHAT1 across all developmental stages and both sexes of S. japonicum (Fig 2A), indicating its essential biological role throughout the parasite’s life cycle. Notably, recombinant SjHAT1 exhibited robust histone acetyltransferase activity, which was inhibited by the compound DW-3–15 in a dose-dependent manner. This pharmacologic property suggests a potential mechanism for the schistosomicidal effects of DW-3–15 against multiple developmental stages of S. japonicum [9–12]. Furthermore, the expression level of SjHAT1 gradually increased as the parasite matured, with significantly higher expression observed in mature female worms compared to their male counterparts of the same age. This sex-biased expression pattern suggests that SjHAT1 may play a critical role in the development and maturity of the female worms. Consistent with the expression data, FISH revealed that SjHAT1 is predominantly localized in the vitellaria of female S. japonicum (Fig 2B). The vitellaria occupy approximately two-thirds of the female body and comprise numerous transversely arranged vitelline lobules extending dorsoventrally. These lobules contain vitelline cells at various developmental stages, which provide both nutrients for fertilized ova and polyphenolic proteins critical for eggshell formation [22,28,30,31]. Mature female schistosomes produce an average of 3,500 eggs per day. Each mature egg consists of one fertilized ovum surrounded by approximately 20 vitelline cells, requiring the female to synthesize 70,000 vitelline cells daily (equivalent to 50 cells per minute) to sustain oviposition [30]. Disruption of vitelline gland function would directly impair egg production. Targeting egg-laying mechanisms not only mitigates immunopathological damage in the host but also represents a strategic intervention for controlling schistosomiasis transmission [28].
Our in vitro experiments further reinforce the potential of SjHAT1 as a promising druggable target for schistosomiasis. Specifically, dsRNA-mediated silencing of SjHAT1 significantly reduced schistosome viability and impaired egg-laying capacity in females, exhibiting time-dependent inhibitory effects. Morphological analysis indicated that SjHAT1-dsRNA impairs the integrity of the female tegument (Fig 4C), a structure critical for schistosome survival [7,23,24]. Furthermore, the tegumental changes observed after SjHAT1 RNA interference closely resembled those induced by DW-3–15. In addition, SjHAT1 knockdown induced notable structural changes in the reproductive organs of S. japonicum adult worms. Following RNA interference, female ovaries displayed severe atrophy and distinct morphological abnormalities, including a marked reduction in mature oocytes, disappearance of intercellular spaces, disorganized cell arrangement, and vacuole formation (Fig 3F). These phenotypic alterations were consistent with those induced by the HAT inhibitor PU139 [18]. A previous study also demonstrated that HAT1 was highly expressed in ovarian granulosa cells (GCs) from young mice and its expression was significantly decreased in aged GCs. siRNA mediated inhibition of HAT1 in GCs decreased the polar body extrusion rate, and increased meiotic defects and aneuploidy in oocytes [32]. Together, these results illustrate the essential role of SjHAT1 in regulating reproductive organ development and egg-laying processes in schistosomes.
Although our in vitro results demonstrated significant anti-schistosomal activity via SjHAT1 knockdown, it is generally acknowledged that this observed efficacy in vitro does not always translate in corresponding parasiticidal effects in vivo [33]. In our case, however, SjHAT1 RNAi in vivo caused a 57.5% reduction (p < 0.0001) in worm burden (Fig 5A) and 65.9% reduction (p < 0.0001) in pairing rate (Fig 5B). Furthermore, the intervention markedly inhibited the developmental progression and maturation of female worms (Fig 5C), with 32.9% arrested at the juvenile stage. This developmental impairment consequently led to compromised oviposition capacity in females. The liver egg deposition was reduced by 79.2% after interference of SjHAT1, which in turn significantly attenuated the pathological damage to the mice (Fig 5F–5H). It is well-known that schistosomes are dioecious, with separate male and female sexes [34]. The sexual development of female schistosomes is entirely dependent on pairing with a male schistosome [35]. In the absence of males, the ovaries and vitellaria of females remain in a primordial state, rendering immature females incapable of laying eggs [36]. Further, it has been shown that physical contact with a male worm, and not insemination, is sufficient to induce female development and the production of viable eggs [37]. Chen et al [38] identified that β-alanyl-tryptamine (BATT), a non-ribosomal peptide produced by male schistosomes is responsible for inducing female sexual development and egg reproduction. Crucially, exogenous BATT alone is sufficient to induce egg laying in immature female parasites [38]. Thus, the reduced male-female interaction, primarily resulting from the decreased male worm population following SjHAT1 knockdown, leads to delayed development of female worms. Additionally, functionally impaired male worms exhibit deficient BATT synthesis, which is essential for triggering maturation and oviposition in females. Moreover, similar phenotypic impairments, including testicular hypoplasia, ovarian degeneration and vitellaria atrophy, were observed in reproductive organs of worms recovered from SjHAT1 RNAi-treated mice in vivo (Fig 5D). These in vivo results demonstrate that SjHAT1 critically regulates the growth, pairing and oviposition in S. japonicum, suggesting that SjHAT1 is a novel druggable target.
Another striking result from our RNA-seq data is the significant downregulation of multidrug resistance-associated protein 4 (MRP4, KSF78_0000799) in females and aromatic L-amino acid decarboxylase (AADC, KSF78_0006508) in males. AADCs are the enzymes catalyzing the decarboxylation of aromatic amino acids (such as tryptophan and tyrosine) to generate monoamines (such as tryptamine and dopamine), which play diverse physiological and biosynthetic roles in living organisms [39]. It has been reported that in planarians, AADC collaborates with nonribosomal peptide synthetase (NRPS) to catalyze the synthesis of BATT [40]. BATT is an essential pheromone that stimulates the maturation of the female reproductive system and promotes egg-laying. Furthermore, in vitro experiments demonstrate that exogenous BATT can replace male worms to induce female reproductive system development and egg production [38]. Knockdown of AADC in sexual planarians resulted in ovary ablation and loss of accessory reproductive organs, including vitellaria, oviducts, sperm ducts, and gonopore [41]. Previous studies have found that AADC is specifically highly expressed in male S. japonicum and is predominantly localized on the inner surface of the gynecophoric canal [42], which exhibits spatial overlap with the distribution of SjHAT1 in male worms. Therefore, we have reason to infer that targeted downregulation of SjHAT1 suppresses the expression of AADC, thereby blocking the synthesis and secretion of the key pheromone BATT in male worms, thus inhibiting the development of the female reproductive system and oviposition. However, how is the BATT signal transduced to female worms? Existing studies have shown that signaling molecules such as linoleic acid, epidermal growth factor, and insulin-like growth factor can enter schistosomes through their excretory system, activating internal signaling pathways [43]. ATP binding cassette (ABC) transporters are the main components of the excretory system. Multidrug resistance-associated proteins (MRPs) are members of ABC transporters. Knockdown of SmMRP1 in S. mansoni adults disrupts egg production and mitigates liver pathology [44]. Our RNA-seq data also revealed a significant downregulation of multidrug resistance-associated protein 4 (MRP4) in female worms, indicating that BATT pheromone might be transduced to females through the MRPs. Taken together, we hypothesize that downregulation of SjHAT1 inhibits the expression of key enzymes such as AADC in male worms, blocking the synthesis and excretion of BATT signals in males; by suppressing the ABC transporter system in female worms, it further hinders BATT signal transduction, thereby suppressing the development of the female reproductive system and oviposition. Verification of this hypothesis could provide new strategies for the prevention and control of schistosomiasis, as well as novel targets for the development of anti-schistosomal drugs.
In conclusion, we have characterized SjHAT1 as a novel anti-schistosomal drug target. It is predominantly expressed in the vitellaria of female worms and in the parenchyma adjacent to the gynecophoral canal of male worms. Knockdown of SjHAT1 impairs worm viability, development and reproduction both in vitro and in vivo. Furthermore, we hypothesize that dual-sex regulatory mechanisms may play a key role in the process of schistosome female egg-laying. Developing specific SjHAT1 inhibitors could yield novel compounds to control schistosomiasis. Future studies on the downstream mechanisms of SjHAT1 will improve our understanding of sexual maturation and oviposition of schistosomes.
Methods
Ethics
All animal experiments were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All efforts were made to relieve the suffering of the experimental animals. The protocol (including mortality aspects) was approved by the Institutional Animal Care and Use Committee (IACUC) of Soochow University (Permit Number: 2007–13).
Animals and parasites
Female C57BL/6 mice (6–8 weeks old, 20.0 ± 5 g) were provided by the Center for Experimental Animals at Soochow University (Suzhou, China). All mice were raised under specific pathogen-free (SPF) conditions with a controlled temperature of 22°C, humidity of 55%, and photoperiod (12 h light, 12 h dark). Each mouse was transcutaneously infected with 60 ± 2 Schistosoma japonicum cercariae (Chinese mainland strain) shedding from snails (Oncomelania hupensis), which were provided by the Institute of Schistosomiasis Control in Jiangsu Province (Wuxi, China). S. japonicum worms were recovered from the infected mice by perfusion through the hepatic portal vein [45]. Three worm pairs were cultured at 37˚C in 5% CO2 in 1 ml m-AB169 medium in a 24-well plate [46]. The medium was changed every other day.
Rapid amplification of cDNA ends (RACE)
Based on the predicted sequences (GenBank: FN318824.1) of SjHAT1 in the NCBI database, an intermediate fragment was amplified by PCR. Subsequently, gene-specific primers (GSP) and nested gene-specific primers (NGSP) were designed from the intermediate fragment by Primer Premier 6 (S4 Table). RACE was performed with SMARTer RACE cDNA Amplification Kit (Clontech, USA) to obtain the terminal sequences. The purified amplification products were directionally cloned into the pRACE vector using the In-Fusion Cloning System. The resultant recombinant plasmids were subjected to bidirectional Sanger sequencing with M13 universal primers, and the obtained sequences were computationally assembled to determine the full-length cDNA of SjHAT1. The open reading frame (ORF) region was analyzed using the ORF Finder tool in the NCBI database, yielding a single complete ORF and its corresponding amino acid sequence.
Multiple sequence alignment and phylogenetic analysis
Homologous proteins of HAT1 across species were retrieved from the UniProtKB/Swiss-Prot database. Multiple sequence alignment was performed with MAFFT v7.520. Putative conserved protein motifs were identified via MEME Suite. The phylogenetic tree construction via the Maximum Likelihood method in MEGA11 with 1000 bootstrap replicates for node support evaluation, and subsequently visualized using Chiplot (https://www.chiplot.online).
Three-Dimensional structure of SjHAT1 and molecular docking analysis with DW-3–15
The three-dimensional structure of SjHAT1 was predicted using AlphaFold3 with default parameters, and the model with the highest confidence score was selected for further analysis [47]. Model reliability was evaluated using predicted local distance difference test (pLDDT), predicted TM-score (pTM), and the predicted aligned error (PAE) matrix. Structural validation was performed using MolProbity, including assessment of MolProbity score, clashscore, and side-chain conformations [48]. Additional validation was conducted using the SAVES v6.1 server, including Ramachandran plot, PROCHECK, ERRAT, and Verify3D analyses, to comprehensively assess the stereochemical quality and structural reliability of the model. The global structural alignment between SjHAT1 and HsHAT1 was performed using the TM-align algorithm under default parameters to calculate the TM-score. Additionally, the Combinatorial Extension (CE) algorithm implemented in PyMOL 3.2 was utilized for local structural superposition. The root-mean-square deviation (RMSD) of Cα atoms was calculated to assess regional conformational differences between the two proteins.
For the DW-3–15/HsHAT1 and DW-3–15/SjHAT1 complexes, a total of 1,000 structural models were generated per complex (25 MSAs × 20 random seeds × 2 samplings). These models were subsequently ranked using the interface-predicted TM-score (ipTM) metric, with the top-100-ranked model from each complex selected for further characterization. Structural predictions were conducted using EnsembleFold [49], a novel framework integrating the CASP15-winning DMFold algorithm [50] with an enhanced AlphaFold3 engine [47]. This integrative approach combines high-fidelity multiple sequence alignment MSA construction with advanced deep learning architectures, enabling accurate modeling of both monomeric and multimeric biomolecular complexes.
Expression and purification of recombinant SjHAT1 (rSjHAT1) in E. coli
The open reading frame (ORF) of SjHAT1 was amplified with forward primer 5’- CGGAATTC ATGGACAGTCTGAATACCAGAA-3’ and reverse primer 5’- CCGCTCGAG TTAAGATTGTTTTTTAAGTGAAGAGACG-3’ using PCR Master Mix (Takara Bio, Shiga, Japan) and cDNA template prepared from adult worms. The PCR product was digested with EcoRI and XhoI (Takara Bio, Shiga, Japan) enzymes and gel-purified, then cloned into expression plasmid pGEX-4T-1. Positive clones were screened and confirmed by DNA sequencing. The restructured plasmid was transformed into Transetta (DE3) (TransGene Biotech Co., Ltd, Bejing, China), and the transformed cells were grown by shaking (220 rpm) in LB medium containing 100 μg/ml ampicillin. Recombinant SjHAT1 was expressed as a glutathione-S-transferase-SjHAT1 fusion protein induced by 0.5 mM isopropy1-β-d-thiogalactoside (IPTG) at 37˚C for 6 h. After induction, the cells were harvested by centrifugation at 5,000 g for 20 min at 4˚C. Add 10 mL pre-cooled PBS containing 1 × Protease inhibitor cocktail for bacterial cell extracts (Beyotime Biotech, Shanghai, China) to1 g of wet bacterial pellet. Vortex the mixture thoroughly to resuspend the cells. Sonicate the suspension on ice using the following parameters: 40% amplitude, 5 s pulses followed by 5 s intervals, for a total duration of 20 min. Centrifuge the lysate at 12,000 rpm for 20 min at 4°C, then collect the supernatant. The supernatant were purified by HyPur T GST 4FF PrePacked Gravity Column (Sangon Biotech, Shanghai, China). Then the protein concentration was determined by a Bradford Protein Assay kit (Beyotime Biotech, Shanghai, China).
Determination of rSjHAT1 enzymatic activity
The enzyme activity of rSjHAT1 was assessed using Histone Acetyltransferase Activity Assay Kit (ab204709, Abcam, USA). Experiments were conducted in duplicate using black flat-bottomed 96-well microplate (Thermo Fisher, Shanghai, China). Standard Curve: standards and enzyme mixtures were prepared to generate standard curve. For each reaction, 50 µL of Histone Acetyltransferase Reaction Mix was prepared as follows: 30 µL HAT assay buffer, 4µL H3 peptide, 10 µL substrate mix, 2 µL Developer, 2 µL PicoProbe, 2 µL Acetyl CoA. Add 50 µL of the reaction mix to the standard (50 µL), sample (50 µL containing 50 ng rSjHAT1), positive control (50 µL) and background (50 µL) wells, followed by incubation at 25°C for 60 minutes. Fluorescence intensity was recorded every 5 min at excitation/emission wavelengths of 535/587 nm. HAT activity was calculated as: HAT activity (µU/mL) = [B/(∆T × V)] × D. Where: B = Amount of CoA from Standard Curve (pmol). ΔT = Reaction time (min). V = Original sample volume added into the reaction well (µL). D = Sample dilution factor. To assess whether DW-3–15-mediated inhibition of SjHAT1, reaction systems containing 50 ng rSjHAT1 were supplemented with DW-3–15 at final concentrations of 0.5, 5, and 50 μM, respectively.
Quantitative real-time PCR (qRT-PCR)
TRIzol Reagent (Invitrogen, USA) was used to extract total RNA from different developmental stages of S. japonicum, including eggs, cercariae, 14-day schistosomula, 21-day, 28-day, and 35-day adult male and female worms. Subsequently, 1 μg total RNA was used for first-strand cDNA synthesis using the RevertAid First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). The cDNA was used as a template for qRT-PCR with ChamQ Universal SYBR qPCR Master Mix (Vazyme, China) and 0.4 µM forward and reverse primers. Amplification was performed on CFX96 Touch Quantitative Real-Time PCR System (Bio-Rad, USA) under standardized conditions: 95°C for 3 min, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, 72°C for 10 s; melt curve analysis: 65°C for 5 s, 95°C for 5 s. The qPCR primers (S4 Table) were designed with Primer Premier 6.0 software and validated through melting curve analysis. Relative quantification was performed using the 2-ΔΔCt method with S. japonicum 26S proteasome non-ATPase regulatory subunit 4 (SjPSMD4, GenBank No. FN320595.1) as the internal reference.
Fluorescence in situ hybridization (FISH)
Males and females recovered from the infected mice at 35 days postinfection (dpi) were fixed in 4% paraformaldehyde at room temperature for 4 h, washed with PBSTX (1 × PBS, 0.3% Triton X-100), and treated with a permeabilization solution (50 mM DTT, 0.1% SDS, 1% NP-40 in 1 × PBS) at 37°C for 10 min. Samples were dehydrated in 100% PBSTX and graded methanol (25%, 50%, 75%, 100%), bleached in 6% bleach solution (30% H2O2 diluted in 100% methanol) under direct light at 4°C for 20 h, and rehydrated in reverse methanol gradients. Rehydrated samples were treated with proteinase K solution (1 μg/ml proteinase K, 0.1% SDS in 1 × PBS) at 37°C for 20 min, re-fixed in 4% paraformaldehyde for 10 min, and incubated with autofluorescence quenchers (Servicebio, China) for 30 min. A CY3-labeled oligonucleotide probe (S4 Table) synthesized by Huzhou Hippo Biotechnology Co., Ltd was hybridized at 56°C for 4 h. Nuclei were counterstained with DAPI, and samples were mounted with anti-fade medium. Visualization was performed on a Leica TCS SP8 Laser Scanning Confocal Microscope (Leica, Germany) using 10 × objective.
Synthesis of dsRNA
The dsRNA templates for SjHAT1-dsRNA and GFP-dsRNA were amplified from the pGEX-4T-1-SjHAT1 maintained in our lab and commercial pET-28a-GFP synthesized by Sangon Biotech (Shanghai, China), respectively, using T7 promoter-containing primers (S4 Table). The size of amplification products was verified by 1% agarose gel electrophoresis, and the identity was confirmed through DNA sequencing. Purified PCR products were employed to synthesis single-stranded RNA using the MEGAshortscript T7 Kit (Invitrogen, USA), following the manufacturer’s protocols. To synthesize dsRNA, a transcription reaction mixture containing equimolar sense and antisense ssRNA strands was denatured initially in a 75 °C water bath for 5 min, followed by annealing at room temperature for 30 min. The resulting dsRNA was subsequently treated with TURBO DNase (Invitrogen, USA) and RNase If (New England Biolabs, USA) to remove residual DNA templates and single-stranded RNA contaminants. After phenol/chloroform extraction and ethanol precipitation, the dsRNA was resuspended in DEPC-treated water. The concentration of dsRNA was quantified using a NanoDrop 2000c Spectrophotometer (Thermo fisher scientific, USA) and adjusted to 3.0 μg/μL. The integrity and size of dsRNA were verified by electrophoresis on 1% agarose gels, and the products were stored at -80 °C.
SjHAT1-RNAi in vitro
S. japonicum paired adults (35 dpi) were obtained and cultured in m-AB169 (1640). Green fluorescent protein (GFP) dsRNA served as the negative control for all RNAi experiments. D1 represents the first day of the experiment. Paired adults were treated with 30 μg/mL dsRNA of GFP or SjHAT1 on D1, D3 and D5 with fresh medium. After daily viability assessments from D1 to D7, worms were collected for stereomicroscopy analysis (Nikon, JPN), and the viability score was assigned as described previously [51], based on the changes in mobility and general appearance. Briefly, each worm was assigned a viability score ranging from 0 to 3. Score 3: worms exhibited the highest activity level as observed in the control group, moving actively and smoothly with transparent bodies; Score 2: worms showed whole-body movement but appeared stiff and slow, with translucent bodies; Score 1: partial movement was observed with opaque body appearance; Score 0: worms remained contracted without resuming movement, considered dead. Concurrently, eggs in the culture medium were collected and enumerated on D3, D5, and D7.
SjHAT1-RNAi in vivo
Nine S. japonicum infected mice were randomly divided into three groups, SjHAT1-dsRNA group, GFP-dsRNA control group, and blank control group, with three mice per group. Ten micrograms of SjHAT1 or GFP dsRNA were injected into the tail vein for interference at 14 dpi, 18 dpi, 22 dpi, 26 dpi, and 30 dpi. An equal volume of 0.7% saline was injected into each mouse in the blank control group at the same time point. Following euthanasia at 35 dpi, adult worms were collected for both RNAi efficacy assessment and phenotypic analysis, while liver tissues were analyzed for egg quantification (see the section “Liver egg counting”) and histopathology (see the section “Histopathological assessment”). The experiments were repeated thrice.
Efficacy of SjHAT1-RNAi
After treatment with SjHAT1 dsRNA, transcriptional levels of SjHAT1 of adult worms were quantified by qRT-PCR performed as previously described. Protein expression levels of SjHAT1 were assessed by Western blotting. To prepare the adult worm soluble protein extracts, phosphate buffer saline precooled on ice containing 20 adult worms was homogenized by sonication (30% power, 5s on, 5s off, 2 min, Q700 Sonicator, USA), and centrifuged for 10min at 13,000 g, 4°C. Protein concentrations were determined by BCA assay. Worm extracts (20 μg protein) were separated on 10% SDS-PAGE gels and transferred to 0.45 μm PVDF membranes (Millipore, Germany). The membranes were blocked with rapid blocking buffer for 15 min at room temperature, then washed with TBST and incubated overnight with rabbit anti-SjHAT1 polyclonal antibody used at 1:500 dilution in TBST at 4°C. After washing with TBST, membranes were incubated for 1 h at room temperature in TBST containing HRP-conjugated goat anti-rabbit IgG (CST, USA) at a dilution of 1:2000. After washing in TBST, the membrane was developed with BeyoECL plus solution (Beyotime Biotech, Shanghai, China) and imaged using Tanon 5200 (Tanon, Shanghai, China).
Morphological observations of schistosomes after interference of SjHAT1
Adult male and female worms were fixed in 4% paraformaldehyde at room temperature overnight, gently compressed between glass slides for 1 h. Specimens were stained with hydrochloric carmine for 2 h, destained in 70% ethanol containing 1% HCl for 20 min, and dehydrated through an ethanol series (80%, 90%, 95%, 100%). After equilibration in ethanol-xylene (1:1) and clearing in 100% xylene, worms were mounted with neutral balsam on glass slides for morphometric and morphological analyses. The morphologies of reproductive organs and germ cells of S. japonicum were observed using a Leica TCS SP8 laser scanning confocal microscope (Leica, Germany) with a 63 × oil immersion objective.
Scanning electron microscopy (SEM)
For SEM analysis, worms were fixed in 1% osmium tetroxide and dehydrated in graded ethanol. After that, the specimens were dried for approximately 30 min. Then the worm samples were mounted on aluminum stubs, coated with gold, and examined with a Hitachi-S4700 scanning electron microscope (Chiyodaku, Japan).
Liver egg counting
0.5g of mouse liver was weighed and incubated overnight in 5% sodium hydroxide (NaOH) at 37°C and 220 rpm to obtain a homogeneous solution. Ten μL of the above solution was observed and counted under an ordinary upright microscope. Each sample was counted 10 times, and the average was calculated. Each group had at least three replicates. The number of liver eggs (EPG) was calculated using the formula: EPG = average×1000/ 0.5
Histopathological assessment
The liver of each mouse was washed with PBS (pH 7.4) and fixed with paraformaldehyde. After dehydration, the tissues were embedded in paraffin. Sections 4-μm-thick were stained with hematoxylin and eosin for granuloma analysis. The areas of granulomas surrounding single egg were observed at 200 × magnification.
Statistics
Data were analyzed with GraphPad Prism or Excel, expressed as means ± SD, and tested for statistical significance using either ANOVA or t-tests, as noted in the figure legends.
RNA sequencing
Worms incubated with SjHAT1- and GFP-dsRNA for 7 days were collected and washed three times with 1 × PBS (pH 7.4). After quick freezing in liquid nitrogen, the samples were sent to SeqHealth Technology Corporation (Wuhan, China) for RNA extraction, library construction and sequencing. RNA integrity was validated using the LabChip GX Touch system (PerkinElmer, USA), followed by fluorometric quantification via Qubit 3.0 Fluorometer (Thermo Fisher Scientific, USA). Libraries were constructed from 500 ng total RNA using the KC mRNA Library Prep Kit (Seqhealth, China) according to the manufacturer’s protocol and sequencing was performed on the DNBSEQ-T7 platform (MGI, China).
Analysis of gene expression profiles and differential expression genes
The FASTQC program (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) performed quality control on Raw data. FASTP v0.23.2 was used to remove low-quality reads and trim adapter sequences to generate clean reads. Clean reads were aligned to the S. japonicum genome (ASM2146165v1) using STAR v2.7.6a, followed by exonic read quantification with featureCounts (Subread-1.5.1) and RPKM normalization for gene expression profiling. Differential expression analysis was performed using the edgeR package (v3.40.2) with thresholds of fold change (FC)>1.5 or ＜1.5 and p < 0.05. Statistically significant GO terms and KEGG pathways enriched in differentially expressed genes (DEGs) were identified using KOBAS v2.1.1 with a significance threshold of p < 0.05. Transcriptional validation of target genes was performed through qRT-PCR as previously described, with primer sequences listed in S4 Table.
Supporting information
S1 Fig. Structural prediction and validation of SjHAT1.
(A) Predicted three-dimensional structure of SjHAT1 color-coded by predicted local distance difference test (pLDDT) values, along with the predicted aligned error (PAE) matrix. Most amino acid residues exhibits pLDDT scores exceeding 80, indicating high reliability at the local structural level. The predicted TM-score (pTM) of 0.86 suggests accurate global folding of the protein. The PAE matrix shows consistently low error values, reflecting stable spatial arrangements between structural domains. (B) Ramachandran plot showing the distribution of backbone dihedral angles. Approximately 91.0% of residues are located in the favored regions of the Ramachandran plot, with none in disallowed regions, indicating excellent stereochemical quality of the protein backbone.
https://doi.org/10.1371/journal.ppat.1014334.s001
(DOCX)
S2 Fig. Efficiency of RNA interference for SjHAT1-dsRNA.
Adult female and male worms with good activity were added to the 24-well plate with five worm per well, different kinds of SjHAT1-dsRNA were added for interference on the first, third, and fifth days. Each group had three replicate wells for each biological experiment, with three biological replicates. DEPC-treated water served as the blank control, and green fluorescent protein (GFP) dsRNA was used as the negative control. (A) Comparison of RNA interference efficiency among SjHAT1-dsRNA1 (30 μg/mL), SjHAT1-dsRNA2 (30 μg/mL), and SjHAT1-dsRNA1/2 (15 μg/mL SjHAT1-dsRNA1 + 15 μg/mL SjHAT1-dsRNA2) in female worms. (B) Comparison of RNA interference efficiency among SjHAT1-dsRNA1, SjHAT1-dsRNA2, and SjHAT1-dsRNA1/2 in male worms. Error bars indicate standard deviation (SD), n = 3 biological replicates, One-Way ANOVA, ‘ns’, not significant, *p < 0.05, **p < 0.01. The raw data is shown in supporting information file [Table I in S1 Data].
https://doi.org/10.1371/journal.ppat.1014334.s002
(DOCX)
S3 Fig. Effect of SjHAT1-dsRNA on worm pairing.
Adults with good pairing activity were incubated in the 24-well plate with three pairs per well. Each group had two replicate wells for each biological experiment, with four biological replicates. (A) Effect of SjHAT1-dsRNA on pairing rate. The raw data is shown in supporting information file [Table J in S1 Data]. (B) Observation under the light microscope on D7 after treatment with GFP-dsRNA and SjHAT1-dsRNA. Error bars indicate standard deviation (SD). *p < 0.01 (Two-Way ANOVA).
https://doi.org/10.1371/journal.ppat.1014334.s003
(DOCX)
S4 Fig. Scanning electron micrographs of the tegument of S. japonicum females in the control group after 3 days incubation.
(A) Gentle panorama of a female worm. (B) Anterior part of the female worm. (C) Mid-portion of the female worm. (D) Spines in the ventral sucker of the female. (E)-(F) Transverse ridges on the tegument of the female worm. Scalebars: A: 1 mm; B: 100 μm; C: 100 μm; D: 5 μm; E: 10 μm; F: 3 μm.
https://doi.org/10.1371/journal.ppat.1014334.s004
(DOCX)
S5 Fig. Scanning electron micrographs of the tegument of female S. japonicum worms after treatment with 100 μM DW-3–15 for 72 h.
(A)The whole worm is swollen and shrunken; (B) extensive sloughing is observed on the tegument in the mid-body of female worm; (C) severe swelling accompanied by numerous blisters is observed at the anterior end of female worm; (D) fusion of the spines in the ventral sucker are obvious; (E)-(F) the ridge-like structures on the tegument of female worm are swollen. Scalebars: A: 1 mm; B: 100 μm; C: 100 μm; D: 5 μm; E: 10 μm; F: 3 μm.
https://doi.org/10.1371/journal.ppat.1014334.s005
(DOCX)
S6 Fig. GFP RNAi has no effect on worm survival, worm pairing and oviposition in vivo.
On the 14th, 18th, 22th, 26th and 30th days postinfection, GFP-dsRNA was injected through the tail vein, and the hepatic portal vein was perfused on the 35th day. (A) Worm burden of the parasites recovered at 35 dpi in Blank and GFP RNAi groups. N = 3 biological replicates, 3 mice of each biological replicates. Student’s t-test, ‘ns’, not significant. The raw data is shown in supporting information file [Table K in S1 Data]. (B) Comparation of the pairing rates of recovered worms between blank control group and GFP RNAi group. N = 3 biological replicates, 3 mice of each biological replicates. Student’s t-test, ‘ns’, not significant. The raw data is shown in supporting information file [Table L in S1 Data]. (C) Observation of the reproductive organs of worms after in vivo treatment. ‘t’, testis; ‘ov’, ovary; ‘vg’, vitelline gland. Scale bars: 20 μm. (D) Gross observations of the mouse liver from the blank and GFP RNAi group. Scale bars: 1 cm. (E) Egg count per gram of liver comparation between the blank and GFP RNAi groups (n = 90), Student’s t-test, ‘ns’, not significant. The raw data is shown in supporting information file [Table M in S1 Data]. (F) Histological assessment of mouse liver by H&E staining. Scale bars: 100 μm. (G) Statistical analysis of the size of egg granuloma area after in vivo treatment (n = 30), Student’s t-test, ‘ns’, not significant. Error bars indicate standard deviation (SD). The raw data is shown in supporting information file [Table N in S1 Data].
https://doi.org/10.1371/journal.ppat.1014334.s006
(DOCX)
S1 Table. Effect of DW-3–15 on the enzymatic activity of recombinant HsHAT1.
*rHsHAT1 activity (pmol/min)=ΔAFU/[time(min)*the slope of the standard curve]. ΔAFU = AFU(sample)-AFU(background). The regression equation of the Standard Curve is Y = 9.7537X + 131.71, R2 = 0.9967. Histone Acetyltransferase Assay Kit (Fluorescent) and rHsHAT1 were purchased from Active Motif (Shanghai, China). The raw data is shown in supporting information file [Table O in S1 Data].
https://doi.org/10.1371/journal.ppat.1014334.s007
(DOCX)
S2 Table. Structural validation statistics of the predicted protein model.
*MolProbity analysis reveals a MolProbity score of 1.52 and a clashscore of 4.38, both characteristic of high-quality structures. Side-chain conformation analysis demonstrates that 98.74% of residues adopt favored rotamer conformations, whereas only 0.25% categorized as poor rotamers. ERRAT analysis yields an overall quality factor of 95.78, indicative of reliable non-bonding atomic interactions. Verify3D analysis confirms that 78.98% of residues have good compatibility between the three-dimensional structure and the amino acid sequence.
https://doi.org/10.1371/journal.ppat.1014334.s008
(DOCX)
S3 Table. The number of mature and immature female worms recovered on 35th day post RNA interference treatment with GFP-dsRNA and SjHAT1-dsRNA.
Chi-square test: χ2(1) =5.528, p = 0.0187.
https://doi.org/10.1371/journal.ppat.1014334.s009
(DOCX)
Acknowledgments
We thank Professor Xing-Quan Zhu for polishing and revising the manuscript. We gratefully acknowledge Professor Zheng Wei and graduate student Yilin Pu of Nankai University for their assistance with the molecular docking analysis of DW-3–15 and HAT1.The infected Oncomelania hupensis snails were provided by the Institute of Schistosomiasis Control in Jiangsu Province (Wuxi, China).
References
- 1. Buonfrate D, Ferrari TCA, Adegnika AA, Russell Stothard J, Gobbi FG. Human schistosomiasis. Lancet. 2025;405(10479):658–70. pmid:39986748
- 2. World Health Organization. Ending the neglect to attain the Sustainable Development Goals: a road map for neglected tropical diseases 2021-2030. Available from: https://www.who.int/publications/i/item/9789240010352
- 3. Wang J, Paz C, Padalino G, Coghlan A, Lu Z, Gradinaru I, et al. Large-scale RNAi screening uncovers therapeutic targets in the parasite Schistosoma mansoni. Science. 2020;369(6511):1649–53. pmid:32973031
- 4. McManus DP, Bergquist R, Cai P, Ranasinghe S, Tebeje BM, You H. Schistosomiasis-from immunopathology to vaccines. Semin Immunopathol. 2020;42(3):355–71. pmid:32076812
- 5. Berger DJ, Park S-K, Crellen T, Vianney TJ, Kabatereine NB, Cotton JA, et al. Extensive transmission and variation in a functional receptor for praziquantel resistance in endemic Schistosoma mansoni. bioRxiv [Preprint]. 2024:2024.08.29.610291. pmid:39257780
- 6. Summers S, Bhattacharyya T, Allan F, Stothard JR, Edielu A, Webster BL. A review of the genetic determinants of praziquantel resistance in Schistosoma mansoni: Is praziquantel and intestinal schistosomiasis a perfect match? Front Trop Dis. 2022;3:933097.
- 7. da Silva VBR, Campos BRKL, de Oliveira JF, Decout J-L, do Carmo Alves de Lima M. Medicinal chemistry of antischistosomal drugs: Praziquantel and oxamniquine. Bioorg Med Chem. 2017;25(13):3259–77. pmid:28495384
- 8. Cupit PM, Cunningham C. What is the mechanism of action of praziquantel and how might resistance strike? Future Med Chem. 2015;7(6):701–5. pmid:25996063
- 9. Duan W, Qiu S, Zhao Y, Sun H, Qiao C, Xia C. Praziquantel derivatives exhibit activity against both juvenile and adult Schistosoma japonicum. Bioorg Med Chem Lett. 2012;22(4):1587–90. pmid:22264473
- 10. Dong L, Duan W, Chen J, Sun H, Qiao C, Xia C. An artemisinin derivative of praziquantel as an orally active antischistosomal agent. PLoS One. 2014;9(11):e112163. pmid:25386745
- 11. Wang X, Yu D, Li C, Zhan T, Zhang T, Ma H, et al. In vitro and in vivo activities of DW-3-15, a commercial praziquantel derivative, against Schistosoma japonicum. Parasit Vectors. 2019;12(1):199. pmid:31053083
- 12. Yang Z-Y, Liu Z-H, Zhang Y-N, Li C, Liu L, Pu W-J, et al. Synergistic effect of combination chemotherapy with praziquantel and DW-3-15 for Schistosoma japonicum in vitro and in vivo. Parasit Vectors. 2021;14(1):550. pmid:34702326
- 13. Xu J, Wang J-Y, Huang P, Liu Z-H, Wang Y-X, Zhang R-Z, et al. Schistosomicidal effects of histone acetyltransferase inhibitors against Schistosoma japonicum juveniles and adult worms in vitro. PLoS Negl Trop Dis. 2024;18(8):e0012428. pmid:39159234
- 14. Fioravanti R, Mautone N, Rovere A, Rotili D, Mai A. Targeting histone acetylation/deacetylation in parasites: an update (2017-2020). Curr Opin Chem Biol. 2020;57:65–74. pmid:32615359
- 15. Park S-Y, Kim J-S. A short guide to histone deacetylases including recent progress on class II enzymes. Exp Mol Med. 2020;52(2):204–12. pmid:32071378
- 16. Hong Y, Cao X, Han Q, Yuan C, Zhang M, Han Y, et al. Proteome-wide analysis of lysine acetylation in adult Schistosoma japonicum worm. J Proteomics. 2016;148:202–12. pmid:27535354
- 17. Li Q, Zhao N, Liu M, Shen H, Huang L, Mo X, et al. Comparative analysis of proteome-wide lysine acetylation in juvenile and adult Schistosoma japonicum. Front Microbiol. 2017;8:2248. pmid:29250037
- 18. Carneiro VC, de Abreu da Silva IC, Torres EJL, Caby S, Lancelot J, Vanderstraete M, et al. Epigenetic changes modulate schistosome egg formation and are a novel target for reducing transmission of schistosomiasis. PLoS Pathog. 2014;10(5):e1004116. pmid:24809504
- 19. Cabezas-Cruz A, Lancelot J, Caby S, Oliveira G, Pierce RJ. Epigenetic control of gene function in schistosomes: a source of therapeutic targets? Front Genet. 2014;5:317. pmid:25309576
- 20. Neuwald AF, Landsman D. GCN5-related histone N-acetyltransferases belong to a diverse superfamily that includes the yeast SPT10 protein. Trends Biochem Sci. 1997;22(5):154–5. pmid:9175471
- 21. Wu H, Moshkina N, Min J, Zeng H, Joshua J, Zhou M-M, et al. Structural basis for substrate specificity and catalysis of human histone acetyltransferase 1. Proc Natl Acad Sci U S A. 2012;109(23):8925–30. pmid:22615379
- 22. Wang J, Collins JJ 3rd. Identification of new markers for the Schistosoma mansoni vitelline lineage. Int J Parasitol. 2016;46(7):405–10. pmid:27056273
- 23. Vale N, Gouveia MJ, Rinaldi G, Brindley PJ, Gärtner F, Correia da Costa JM. Praziquantel for schistosomiasis: single-drug metabolism revisited, mode of action, and resistance. Antimicrob Agents Chemother. 2017;61(5):e02582-16.
- 24. Xu J, Dong L-L, Sun H, Huang P, Zhang R-Z, Wang X-Y, et al. Small change, big difference: A promising praziquantel derivative designated P96 with broad-spectrum antischistosomal activity for chemotherapy of schistosomiasis japonica. PLoS Negl Trop Dis. 2023;17(7):e0011215. pmid:37410790
- 25. Popiel I. Male-stimulated female maturation in Schistosoma: a review. J Chem Ecol. 1986;12(8):1745–54. pmid:24305892
- 26. Kunz W. Schistosome male-female interaction: induction of germ-cell differentiation. Trends Parasitol. 2001;17(5):227–31. pmid:11323306
- 27. LoVerde PT. Presidential address. Sex and schistosomes: an interesting biological interplay with control implications. J Parasitol. 2002;88(1):3–13. pmid:12053976
- 28. Sun C, Luo F, You Y, Gu M, Yang W, Yi C, et al. MicroRNA-1 targets ribosomal protein genes to regulate the growth, development and reproduction of Schistosoma japonicum. Int J Parasitol. 2023;53(11–12):637–49. pmid:37355197
- 29. Xiao SH, Yue WJ, Yang YQ, You JQ. Susceptibility of Schistosoma japonicum to different developmental stages to praziquantel. Chin Med J (Engl). 1987;100(9):759–68. pmid:3127152
- 30. He Y, Yang H. Physiology of egg formation of Schistosoma japonicum (in Chinese). Acta Zool Sin. 1974;20(3):243–62.
- 31. Moore DV, Sandground JH. The relative egg producing capacity of Schistosoma mansoni and Schistosoma japonicum. Am J Trop Med Hyg. 1956;5(5):831–40.
- 32. Chen M, Liu Y, Liu Z, Su L, Yan L, Huang Y, et al. Histone acetyltransferase Gcn5-mediated histone H3 acetylation facilitates cryptococcal morphogenesis and sexual reproduction. mSphere. 2023;8(6):e0029923. pmid:37850793
- 33. Patra M, Ingram K, Pierroz V, Ferrari S, Spingler B, Gasser RB, et al. [(η(6)-Praziquantel)Cr(CO)3] derivatives with remarkable in vitro anti-schistosomal activity. Chemistry. 2013;19(7):2232–5. pmid:23296750
- 34. Cort WW. Sex in the trematode family Schistosomidae. Science. 1921;53(1367):226–8. pmid:17734017
- 35. Severinghaus AE. Sex studies of Schistosoma japonicum. J Cell Sci. 1928;S2-71(284):653–702.
- 36. Clough ER. Morphology and reproductive organs and oogenesis in bisexual and unisexual transplants of mature Schistosoma mansoni females. J Parasitol. 1981;67(4):535–9. pmid:7264839
- 37. Wang J, Chen R, Collins JJ 3rd. Systematically improved in vitro culture conditions reveal new insights into the reproductive biology of the human parasite Schistosoma mansoni. PLoS Biol. 2019;17(5):e3000254. pmid:31067225
- 38. Chen R, Wang J, Gradinaru I, Vu HS, Geboers S, Naidoo J, et al. A male-derived nonribosomal peptide pheromone controls female schistosome development. Cell. 2022;185(9):1506-1520.e17. pmid:35385687
- 39. Han S-W, Shin J-S. Aromatic L-amino acid decarboxylases: mechanistic features and microbial applications. Appl Microbiol Biotechnol. 2022;106(12):4445–58. pmid:35763068
- 40. Issigonis M, Browder KL, Chen R, Collins JJ 3rd, Newmark PA. A niche-derived nonribosomal peptide triggers planarian sexual development. Proc Natl Acad Sci U S A. 2024;121(26):e2321349121. pmid:38889152
- 41. Khan UW, Newmark PA. Somatic regulation of female germ cell regeneration and development in planarians. Cell Rep. 2022;38(11):110525. pmid:35294875
- 42. Wang J, Yu Y, Shen H, Qing T, Zheng Y, Li Q, et al. Dynamic transcriptomes identify biogenic amines and insect-like hormonal regulation for mediating reproduction in Schistosoma japonicum. Nat Commun. 2017;8:14693. pmid:28287085
- 43. Kusel JR, McVeigh P, Thornhill JA. The schistosome excretory system: a key to regulation of metabolism, drug excretion and host interaction. Trends Parasitol. 2009;25(8):353–8. pmid:19617001
- 44. Kasinathan RS, Morgan WM, Greenberg RM. Genetic knockdown and pharmacological inhibition of parasite multidrug resistance transporters disrupts egg production in Schistosoma mansoni. PLoS Negl Trop Dis. 2011;5(12):e1425. pmid:22163059
- 45. Duvall RH, DeWitt WB. An improved perfusion technique for recovering adult schistosomes from laboratory animals. Am J Trop Med Hyg. 1967;16(4):483–6. pmid:4952149
- 46. You Y, Chen X, Huo L, Chen L, Chen G, Gu M, et al. An improved medium for in vitro studies of female reproduction and oviposition in Schistosoma japonicum. Parasit Vectors. 2024;17(1):116. pmid:38454463
- 47. Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature. 2024;630(8016):493–500. pmid:38718835
- 48. Chen VB, Arendall WB 3rd, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 1):12–21. pmid:20057044
- 49. Wuyun Q, Liu Q, Ni W, Peng C, Zhang Z, Zhou X, et al. Alternative conformation prediction using deep learning with multi-MSA strategy and structural clustering in CASP16. Proteins. 2026;94(1):348–61. pmid:41014267
- 50. Zheng W, Wuyun Q, Li Y, Zhang C, Freddolino L, Zhang Y. Improving deep learning protein monomer and complex structure prediction using DeepMSA2 with huge metagenomics data. Nat Methods. 2024;21(2):279–89. pmid:38167654
- 51. Santiago E de F, de Oliveira SA, de Oliveira Filho GB, Moreira DRM, Gomes PAT, da Silva AL, et al. Evaluation of the anti-Schistosoma mansoni activity of thiosemicarbazones and thiazoles. Antimicrob Agents Chemother. 2014;58(1):352–63. pmid:24165185