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. 2026 Jul 29;125(1):136. doi: 10.1007/s00436-026-08737-8

Lethal effect of trehalose-6-Phosphate phosphatase inhibitor N-(phenylthio) phthalimide on Trichinella spiralis

Jiaming Hou 1,#, Rulin Ruan 1,#, Ting Zhang 1, Yongqing Yang 1, Han Xue 1, Mingxin Song 1,✉, Caixia Han 1,✉
PMCID: PMC13714049  PMID: 42944908

Abstract

In eukaryotes, with the exception of mammals, trehalose fulfills multiple biological functions, including energy provision and stress tolerance. Trehalose-6-phosphate phosphatase (TPP) is a key enzyme in the trehalose biosynthetic pathway, rather than a trehalose synthase per se. Loss of TPP function or knockdown of its expression impairs trehalose synthesis, which in turn leads to developmental abnormalities or lethality in nematodes. These observations suggest that TPP represents a potential target for the development of anti-parasitic therapeutics. This study focused on the cloning, expression, and characterization of Trichinella spiralis TPP (TsTPP). A recombinant plasmid was constructed to express and purify the recombinant TsTPP protein (rTsTPP), whose immunogenicity was confirmed using positive serum from Trichinella spiralis (T. spiralis)-infected mouse. The expression of TsTPP at different developmental stages of T. spiralis was determined by immunofluorescence assay. The specific TPP inhibitor N-(phenylthio) phthalimide (NPP) was employed to evaluate its lethal effect on T. spiralis muscle larvae (ML) in vitro. Furthermore, molecular methods such as qPCR and Western blot were used to detect the transcription and protein expression levels of TsTPP upon NPP treatment, aiming to identify new drug targets against T. spiralis. We successfully constructed the recombinant plasmid and obtained rTsTPP. Positive serum assays demonstrated the immunogenicity of rTsTPP, and we used indirect immunofluorescence assay (IFA) to determine its localization across different developmental stages of T. spiralis. Distinct distribution patterns were observed: TsTPP was diffusely distributed throughout the body of newborn larvae (NBL), accumulated in the intestinal glands, midgut ampulla and genital primordia of muscle larvae (ML), and localized to the mid and posterior intestine of adult worms (AW). To verify the lethal effect of NPP in vitro, we treated ML with NPP at concentrations of 12.5, 25, 50, 100, and 200 µg/mL for 24, 36, and 48 h, and evaluated its lethal effect by statistical analysis of worm morphology. The experimental results showed that the mortality rate of ML gradually increased with increasing NPP concentration and culture time. When the concentration was 200 µg/mL, the survival rate of ML was less than 50%. Furthermore, when treated with NPP at concentrations of 12.5, 25, 50, 100, and 200 µg/mL for 24 h, the transcription levels of the TsTPP gene decreased by 19%, 23%, 30%, 38% (all p < 0.05), and 57% (p < 0.01), respectively; after 36 h of treatment, the transcription levels of TsTPP gene were reduced by 18% (p < 0.05), 23%, 35%, 39%, and 60% (all p < 0.01); after 48 h of treatment, the transcription levels of TsTPP gene decreased by 19%, 23%, 42%, 42%, and 65% (all p < 0.01), respectively. N-(phenylthio) phthalimide (NPP) exhibits significant lethal activity against T. spiralis by inhibiting the trehalose synthesis pathway, providing basic data for future NPP as a new clinical drug target.

Keywords: Trichinella spiralis, Trehalose-6-phosphate phosphatase, N-(phenylthiol)phthalimide, antiparasitic target

Background

Trichinellosis is a foodborne zoonotic parasitic disease affecting both humans and animals (Alban et al. 2024, Jiang et al. 2016). When a host ingests muscle tissue containing infective muscle larvae (ML) of T. spiralis, gastric digestion causes the ML to be released from their cysts. Stimulated by bile, the ML activate into intestinal infective larvae (IIL), migrating to the small intestine within hours and invading columnar epithelial cells. After four molts, they ultimately develop into adult worms (AW) (Ferguson and Castro 1973, Romarís and Appleton 2001). Following mating, the female AW releases newborn larvae (NBL) (Despommier 1998). T. spiralis infection can cause a wide range of clinical manifestations, from gastrointestinal symptoms to fever and myalgia. Severe cases may result in death due to complications such as myocarditis, heart failure, or neurological lesions (Compton et al. 1993, Lopez-Lozano et al. 1988). Therefore, developing vaccines or identifying drug targets is of great importance for the prevention and treatment of trichinellosis (Xu et al. 2022, Zhang et al. 2022).

Trehalose is a natural non-reducing disaccharide widely present in various microorganisms, fungi, plants, nematodes, insects, and certain invertebrates, but it is absent in mammals (Jain and Roy 2009, Avonce et al. 2006). Trehalose protects cellular structures from multiple environmental stresses, such as drought (Liu et al. 2024), freezing (Zhong et al. 2024), and osmotic pressure (Wang et al. 2024). In nature, the biosynthetic pathways of trehalose encompass five distinct routes: the Trehalose-6-phosphate synthase–trehalose-6-phosphate phosphatase (TPS-TPP) pathway, the Maltooligosyl trehalose synthase–maltooligosyl trehalose trehalohydrolase (TreY-TreZ) pathway, the Trehalose synthase (TreS) pathway, the Trehalose glycosyltransferring synthase (TreT) pathway, and the Trehalose phosphorylase (TreP) pathway. However, eukaryotes possess only the TPS-TPP pathway, which involves trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) (Avonce et al. 2006). First, TPS forms the intermediate product trehalose-6-phosphate (T6P), which is subsequently dephosphorylated by TPP to yield trehalose (Paul et al. 2020). Several studies have highlighted the involvement of TPP in parasite survival, environmental adaptation, and infectivity. In Caenorhabditis elegans, loss of function in the gob-1 gene encoding TPP causes intestinal obstruction, leading to larval mortality (Kormish and McGhee 2005). Concurrently, TPS and TPP activities have been detected in the muscle, reproductive system, and hemolymph of Ascaris suum(Dmitryjuk et al. 2009). Notably, in Brugia malayi, RNAi-mediated silencing of the TPP gene resulted in embryonic malformations or developmental arrest and caused mortality in infective larvae (L3). When L3 surviving 24 h after TPP gene silencing were inoculated into the peritoneal cavity of jirds (Meriones unguiculatus), an 84.9% reduction in worm numbers was observed (Kushwaha et al. 2012), validating TPP as a potential drug target for filariasis control. Therefore, a study has included the TPP of B. malayi in the priority list of anticipated drug targets for lymphatic filariasis and constructed a vaccine model against it (Singh et al. 2025).

Phthalimide derivatives exhibit diverse biological activities and hold significant applications in antitumor (Spallarossa et al. 2009, Kok et al. 2008), anti-inflammatory (Machado et al. 2005, Lima et al. 2002), and anti-angiogenic (Noguchi et al. 2005) contexts. Following C4-site functionalization of the phthalimide compound thalidomide, its derivatives exhibited significant α-glucosidase inhibitory activity, demonstrating substantially enhanced efficacy compared to the parent compound. This discovery provides a chemical foundation for developing anti-parasitic agents targeting glucose-metabolizing enzymes (Motoshima et al. 2009). Subsequently, Cross et al. (Cross et al. 2019) screened a compound library and identified N-(Phenylthio)phthalimide (NPP), evaluating its inhibitory effect on TPP activity in four nematodes: Ancylostoma ceylanicum, Toxocara canis, among others. Results demonstrated that NPP inhibited TPP activity in all these nematodes. Additionally, NPP reduced adult nematode survival rates and larval migration rates.

Based on this, screening for target protein inhibitors holds promise as an effective strategy in the development of anti-T. spiralis agents. Therefore, this study aims to investigate the lethal effects and parasiticidal mechanisms of the TPP inhibitor NPP against T. spiralis in vitro. This research seeks to further elucidate the role of trehalose in the growth and development of T. spiralis, thereby providing a new direction for exploring novel anti-T. spiralis agent development.

Methods and methods

Trichinella strain and experimental animals

The T. spiralis strain (ISS533) was originally isolated from a swine in Heilongjiang Province, China, and has been maintained and passaged in Kunming mice by the Department of Parasitology, College of Veterinary Medicine, Northeast Agricultural University. Six-week-old specific pathogen-free (SPF) female Kunming mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. Female New Zealand white rabbits, weighing approximately 3.0 kg, were purchased from the Experimental Animal Center of Harbin Medical University. T. spiralis ML were isolated from mice 42 days post-infection using the Baermann method (Jiang et al. 2012). NBL and AW were collected from the small intestines of infected mice at different stages post-infection (Liu et al. 2015, Wu et al. 2016). All experimental procedures were approved by the Animal Management Committee of Northeast Agricultural University and performed in accordance with animal ethics guidelines and the approved protocol (Animal Ethics Committee Approval No.: NEAUEC 20210334).

Bioinformatic analysis of TsTPP

Referring to the TsTPP gene sequence (KRY30566.1) published on NCBI, the characteristics of TsTPP were predicted using bioinformatics software and web servers. Transmembrane domains and signal peptides were predicted using TMHMM and SignalP4.1, respectively, while protein domains were predicted using NCBI.

Acquisition of cDNA from T. spiralis ML

T. spiralis ML were lysed using TRIzol reagent (Takara, Dalian, China). The larvae were completely homogenized by ultrasonication with a cycle of 3 s on and 3 s off for 3 min using ultrasonic disruptor (JY88-IIN, SCIENTZ). Total RNA was then extracted and reverse transcribed into cDNA (TransScript® One-Step RT-PCR SuperMix, TransGen). The resulting cDNA product was stored at -20 °C for subsequent use.

Construction of plasmids and preparation of polyclonal antibodies

The TsTPP gene was PCR amplified using the primers carrying the restriction enzyme sites BamHI and HindIII (5’-CGAGCTCATGTCCATGCGTTTGCTAGTTGTCTTC A-3’; 5’-CCCAAGCTTCTACGTGGTAAGGGCACCGCATTTG-3’). Briefly, PCR amplification of the target gene was performed using the KOD Plus Neo high-fidelity DNA polymerase (1.0 U/µL) in a 20 µL reaction system containing 14.0 µL ddH₂O, 1.0 µL KOD Plus Neo, 2.5 µL dNTP mixture, 2.0 µL MgSO₄, 1.0 µL forward primer (10 µM), 1.0 µL reverse primer (10 µM), 1.0 µL cDNA template, and 2.5 µL 10× reaction buffer. The thermal cycling protocol consisted of an initial denaturation at 94 °C for 5 min, followed by 36 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 68 °C for 45 s, with a final extension at 72 °C for 5 min and an indefinite hold at 4 °C. The 1419-bp PCR products were cloned into the vector pET30a(+) and the recombinant pET30a-TsTPP plasmid was tansformed into Escherichia coli BL21 (DE3). Plasmid DNA was extracted and subjected to double digestion with BamHI and HindIII, followed by electrophoretic verification of the digested products. Protein was achieved using a Ni-NTA resin column, and its immunogenicity was detected using positive serum of T. spiralis by Western blot method (Gao et al. 2021).

New Zealand White rabbits were immunized with 1 mg rTsTPP emulsified with Incomplete Freund’s Adjuvant, administered through three rounds of multipoint subcutaneous injections along the back. The intervals were 7 days after the first immunization and 14 days after the second immunization. Blood was collected from the marginal ear vein before immunization and 7 days after the final immunization to obtain negative control serum and positive antiserum, respectively. The titer of the polyclonal antibodies was determined by indirect ELISA.

Analysis of immunoreactivity

Purified rTsTPP and ML total protein samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk at 37 °C for 2 h. Subsequently, they were incubated overnight at 4 °C with the primary antibodies (a 1:500 dilution of positive serum from mice infected with T. spiralis for 42 days and a 1:1000 dilution of anti-rTsTPP polyclonal rabbit antiserum). After being washed with TBST (1% Twain-20), the membranes were further incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:8000, Solarbio, Beijing, China) and HRP-conjugated goat anti-rabbit IgG (1:6000, Solarbio, Beijing, China), respectively. Eventually, the reaction was detected using the Genesys detection system (Syngene, Cambridge, UK) and analyzed using Image J.

Rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:1000, TransGen Biotech, Beijing, China) was used as an internal reference to analyze the expression levels of TsTPP in T. spiralis ML treated with different concentrations of NPP.

Immunofluorescence test of Tissue sections

Sections were prepared from T. spiralis at three developmental stages: NBL, ML, and AW. The sections were first blocked with 1 ml of goat serum at 37 ℃ for 1 h. After washing, they were incubated overnight at 4 °C with rabbit anti-rTsTPP serum (1:400). Following another wash, the sections were incubated with a FITC-conjugated goat anti-rabbit IgG (H + L) (1:1000; TransGen Biotech, Beijing, China) at 37 °C for 1 h. Finally, the sections were mounted with an antifade mounting medium to preserve fluorescence and imaged using a laser scanning confocal microscope (Gao et al. 2021).

Treatment of T. spiralis ML in vitro

N-(phenylthio) phthalimide (NPP) (Aladdin, Shanghai, China) was dissolved in 10% dimethyl sulfoxide (DMSO) (Aladdin, Shanghai, China) to prepare the stock solution. The final concentrations of NPP were set at 0, 12.5, 25, 50, 100, and 200 µg/mL, while the control group contained only 8% DMSO. Trichinella spiralis ML were cultured in 12-well plates. Each well contained 1000 ML in 1 mL of RPMI-1640 medium (MeilunBio, Dalian, China) supplemented with 1% penicillin-streptomycin (Solarbio, Beijing, China). Each experimental condition was performed in triplicate. The plates were incubated at 37 ℃ under 5% CO₂ for 24, 36, or 48 h. After incubation, ML from each well were collected and examined under a microscope to calculate the mortality rate. ML were considered dead if they exhibited an uncoiled morphology and showed no movement for over 30 s. The number of surviving ML was counted, and the mortality rate was calculated using the following formula: Mortality (%) = [(Total ML - Surviving ML) / Total ML] × 100%.

qPCR analysis of GAPDH gene expression in NTT-treated T. spiralis larvae

Specific primers for TsTPP and TsGAPDH (internal reference) were designed based on sequences obtained from the NCBI database. The primer sequences were as follows: TsTPP-F: 5’-AGCTGAAAGATCTGGCCACC-3’; TsTPP-R: 5’-TCCAAGTCCCCT GTGTGGTA-3’; TsGAPDH-F: 5’-TGGCTTAGCTCCGTTGG-3’; TsGAPDH-R: 5’-TTTGGGTTGCCGTTGTA-3’. qPCR was performed using a LightCycler® 480 system (Roche, Germany) to assess the transcriptional level of the TsTPP gene. Relative gene transcriptional level was calculated using the 2−ΔΔCt method.

Determination of TPP enzymatic activity and trehalose content in T. spiralis ML

Parasite tissues were homogenized with normal saline (1:10, v/v), disrupted, and centrifuged at 3000 rpm for 10 min at 4 °C; the supernatant was collected, and its total protein concentration was determined. Trehalose content was measured via the anthrone colorimetric method: briefly, muscle larvae were homogenized in extraction solution, incubated, centrifuged at 8000 g, and the supernatant was reacted with anthrone–sulfuric acid working solution (0.2% anthrone in concentrated H₂SO₄) at 95 °C before measuring absorbance at 630 nm for calculation against a standard curve. Trehalose-6-phosphate phosphatase activity was assayed using a commercial kit following the manufacturer’s instructions and trehalose content (Suzhou Grace Biotechnology Co.,Ltd).

Statistical analysis

The data were obtained from three independent biological replicates. All statistical analyses were performed using GraphPad Prism 8.0. Quantitative results are presented as mean ± standard deviation (SD). For the mortality data involving two factors, two-way analysis of variance (two-way ANOVA) was applied to evaluate the main effects of drug concentration, incubation time, and their interaction effect. Following two-way ANOVA, Tukey’s multiple comparisons test was adopted for post-hoc pairwise comparisons. Statistical significance was defined as P < 0.05, and P < 0.01 was considered highly significant.

Results

Bioinformatics analysis of TsTPP gene sequences

According to the NCBI database and protein relative size prediction website (https://www.novopro.cn/tools/protein_mw.html), it was evident that the complete TsTPP cDNA sequence was 1419 bp encoding a protein of 472 amino acids, with a molecular weight (MW) of 53.18 kDa. The prediction results of TMHMM and SignalP 4.1 Server revealed that TsTPP contains a transmembrane domain located at positions 5–27 (Fig. 1A) and no signal peptide (Fig. 1B). Protein domain prediction using NCBI identified the presence of a TPP domain and a Cof domain in this protein (Fig. 1C).

Fig. 1.

Fig. 1

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Bioinformatics analysis of TsTPP

(A) transmembrane domain prediction of TsTPP; (B) signal peptide prediction of TsTPP; (C) protein domain prediction of TsTPP

Directional cloning of the TsTPP gene in pET30a(+) vector

The TsTPP gene was successfully amplified from the cDNA pool of T. spiralis ML, yielding products of 1419 bp (Fig. 2A). Sequence alignment of the cloned gene was performed using DNAMAN 10 software, and the results showed no mutation in amplified gene products (Fig. 2B). TsTPP gene was subsequently cloned successfully in pET30a(+) as confirmed by restriction digestion of recombinant construct of pET30a-TPP (Fig. 2C).

Fig. 2.

Fig. 2

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Cloning and validation of TsTPP gene in pET30a(+) vector

(A) PCR amplification of TsTPP. M: 2000 bp DNA marker; lane 1: PCR products of TsTPP. (B) Sequence alignment of TsTPP with amplified gene products. (C) Confirmation of TsTPP insert in pET30a construct: cloning into pET30a was verified by digesting the plasmid construct with restriction enzymes BamHI and HindIII. M: 2000 bp DNA marker; lane 1: undigested plasmid; lane 2: BamHI and Hind III digested pET30a-TPP plasmid construct

Immunoflurosence assay for the analysis of expression and localization of rTsTPP

The recombinant protein of approximately 53 kDa was expressed after induction, and Western blot analysis confirmed specific binding of rTsTPP with positive serum of T. spiralis (Fig. 3A). ELISA detection showed that the titer of the polyclonal antiserum against rTsTPP reached 1:2560 (Fig. 3B). Immunofluorescent localization revealed a dynamic spatiotemporal distribution pattern of the protein in T. spiralis: it was distributed throughout the worms in NBL, became enriched in the intestinal glands, midgut ampulla, and genital primordia in ML, and was localized to the mid-to-hindgut in AW. These results indicate that TsTPP is present throughout the entire life cycle of T. spiralis (Fig. 3C).

Fig. 3.

Fig. 3

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Expression and immunofluorescent localization of rTsTPP

(A) Western blotting of the rTsTPP. M: protein marker; lane 1: rTsTPP was probed with mouse infection serum; 2: rTsTPP was probed with healthy mouse serum. (B) Analysis of polyclonal antibody titer; (C) Immunofluorescence localization analysis of TsTPP. Scale bars of NBLs and AWs = 1000 μm; Scale bars of MLs = 100 μm

In vitro analysis of the nematicidal activity of NPP against T. spiralis ML

The results showed that the mortality rate of T. spiralis ML increased significantly and continuously with rising concentrations of NPP and longer exposure times, respectively. When NPP concentrations ranged from 12.5 to 50 µg/mL, the ML mortality rates were 25.62%-39.12% after 24 h of exposure, 21.88%-46.62% after 36 h, and 29.65%-54.64% after 48 h. At an NPP concentration of 200 µg/mL, the mortality rate increased from 56.19% to 66.72% as the exposure time was extended (Fig. 4). The estimated lethal concentrations (LC) to kill 50% (LC50) tended to decrease over the observation period from 24 to 48 h, from 133.7 µg/mL to 48.45 µg/mL. The LC₅₀ value at 24 h was 2.8 fold higher than that at 48 h (Table 1).

Fig. 4.

Fig. 4

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Mortality rates of T. spiralis ML exposed to increasing concentrations of N-(phenylthiol) phthalimide from 24 to 48 h

Table 1.

Lethal concentration of NPP to T. spiralis ML from 24 to 48 h

Time (h) LC50 µg/mL LC50 (LCI to UCI) µg/mL
24 133.7 101.8 ~ 195.7
36 75.26 58.71 ~ 100.7
48 48.45 38.22 ~ 61.37
LC, lethal concentration; LCI, lower confidence interval; UCI, upper confidence interval.
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Detection of protein expression level post NPP treatment

The transcriptional level of TsTPP mRNA was detected by qPCR. The results showed that with increasing concentrations of NPP and longer exposure times, the transcriptional level of the TsTPP mRNA exhibited an overall dose- and time-dependent decrease. After 48 h of in vitro culture, even at a low NPP concentration of 12.5 µg/mL, the transcriptional level of the TsTPP mRNA was already significantly reduced (P < 0.01). When the concentration of NPP reached 200 µg/mL, the transcription levels of TsTPP mRNA significantly decreased at 24, 36, and 48 h, respectively, to 43.24%, 40.18%, and 35.37% of the control group, respectively (Figure. 5 A). Under the same NPP concentration, the effect was more pronounced after 48 h of treatment. Therefore, 48 h was selected as the treatment time point for subsequent experiments.

Use Image J software to analyze the expression level of TsTPP in western blot results. Western blot results showed that after 48 h of NPP exposure, the expression level of TsTPP protein in ML decreased overall, declining to 61.02%, 54.12%, 32.22%, 41.89%, and 36.63% of the control group, respectively (P > 0.05, P < 0.05, P < 0.01, P < 0.01, P < 0.01, respectively) (Fig. 5B and C).

Fig. 5.

Fig. 5

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TsTPP mRNA transcription and protein expression in T. spiralis ML

(A)The transcription of TsTPP mRNA in T. spiralis was incubated in different concentrations of NPP for 24, 36 and 48 h. (B, C) The expression of TsTPP protein in T. spiralis was incubated in different concentrations of NPP for 48 h. * indicates that the difference is significant (P < 0.05). ** or *** indicates that the difference is extremely significant (P < 0.01 or P < 0.001)

Determination of TPP enzymatic activity and trehalose content in T. spiralis ML

T. spiralis ML were treated with different concentrations of NPP to examine its effects on TsTPP enzymatic activity and trehalose content. The results showed slightly increased TsTPP activity in comparison to the control group but not significantly at NPP concentrations of 12.5 µg/mL and 25 µg/mL (P > 0.05). As the NPP concentration continued to rise, TsTPP enzymatic activity exhibited a significant decreasing trend in a concentration‑dependent pattern (Fig. 6A). Additionally, as the NPP concentration increased, the trehalose content in the larvae initially showed a slight increase and then decreased. When the NPP concentration reached 50 µg/mL or higher, the trehalose content was significantly lower than that in the control group (Fig. 6B), a trend largely consistent with that observed for TPP enzymatic activity.

Fig. 6.

Fig. 6

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Enzymatic activity of TsTPP and content of trehalose in T. spiralis ML

(A) Enzymatic activity of TsTPP in T. spiralis ML was incubated in different concentrations of NPP for 48 h. (B) Trehalose content in T. spiralis ML was incubated in different concentrations of NPP for 48 h. * indicates that the difference is significant (P < 0.05). ** or *** indicates that the difference is extremely significant (P < 0.01 or P < 0.001)

Discussion

Trehalose is not only utilized as source of energy in organisms (CANDY and KILBY 1961, Elbein et al. 2003), but also protects them from external stress, enabling survival under harsh conditions (Eh et al. 2025, Shrestha et al. 2024). Trehalose-6-phosphate phosphatase (TPP), as a conserved metabolic enzyme that catalyzes trehalose formation, is highly conserved in pathogens and lacks a homologous pathways in mammals (Jain and Roy 2009, Wang et al. 2024). The trehalose synthesis pathway may serve as a potential target for anthelmintics (Łopieńska-Biernat et al. 2018). Therefore, this study focuses on investigating the trehalose synthesis pathway in T. spiralis as a target for anti-parasitic agents.

In this study, the rTsTPP protein was successfully expressed. The purified rTsTPP exhibited immunogenicity and was capable of inducing anti-rTsTPP antibodies, confirming that T. spiralis has the same trehalose synthesis pathway as like other nematodes such as C. elegans (Kormish and McGhee 2005), (A) suum (Dmitryjuk et al. 2009), and (B) malayi

(Kushwaha et al. 2011). Since TsTPP, a key enzyme in the trehalose synthesis pathway, is homologous to proteins in other nematodes and can induce specific antibodies, this suggests that T. spiralis also possesses the same trehalose synthesis pathway as these nematodes. Immunofluorescence localization demonstrated that TsTPP is present throughout all stages of T. spiralis, confirming its fundamental role in energy supply during growth and development. However, its expression level and localization varied across different stages. In NBL, which corresponds to the active invasive phase, TsTPP was diffusely distributed throughout the worms, suggesting it may provide extensive energy support for the worms’ rapid movement and initial parasitism. During the encystment stage, TsTPP became enriched in specific regions such as the intestinal glands, midgut ampulla, and genital primordia. This shift implies that the trehalose synthesis mediated by TsTPP may not only meet basic energy demands but also be more specifically involved in supporting physiological activities related to parasitism and reproduction. In AW, TsTPP was localized in the mid and posterior intestine, which aligns with the adult worms’ adaptation to residing in the host intestine and relying on the absorption and digestion of host nutrients, potentially participating in local glucose metabolism regulation and energy supply in the intestine.

As an inhibitor of nematode TPP, NPP shares spatial and topological similarity with T6P, the substrate of TPP. N-(phenylthio) phthalimide (NPP) can occupy the TPP binding site and subsequently interact with TPP’s conserved cysteine residues under non-reducing conditions, thereby acting as a parasiticidal inhibitor (Cross et al. 2019). Another study indicated that NPP inhibited TPP activity in lepidopteran insects (Helicoverpa armigera), disrupted trehalose catabolism, and consequently regulated energy storage, ultimately leading to reduced larval size, weight, and pupation rates (Tellis et al. 2024). Thus, NPP can effectively inhibit TPP enzymatic activity and exhibits favorable insecticidal efficacy.

To further investigate the biological functions of TsTPP, we treated T. spiralis ML in vitro with its specific inhibitor, NPP. Consistent with the aforementioned studies, we found that the mortality rate of the worms increased significantly with increasing NPP concentrations and longer exposure times. Further investigation showed that NPP inhibited both the mRNA and protein expression levels of TsTPP; furthermore, NPP also reduced TPP enzymatic activity and trehalose content within the worms. These results indicate that NPP blocks the conversion of T6P into trehalose by inhibiting TPP enzymatic activity. The subsequent decline in trehalose content may, on one hand, lead to a shortage of glucose supply (Tellis et al. 2024), thereby confronting the worms with an energy crisis. On the other hand, as a classical stress-protective molecule (Liu et al. 2024, Zhong et al. 2024, Wang et al. 2024), the depletion of trehalose could significantly weaken the worms’ resistance to internal and external stresses, such as oxidative stress and pH fluctuations (Wang et al. 2023), ultimately leading to worm death. In contrast, the slight increases in TPP enzymatic activity and trehalose content observed at NPP concentrations of 12.5 µg/mL and 25 µg/mL might be attributed to a stress response triggered in T. spiralis ML upon exposure to lower NPP concentrations. The alteration of the optimal living conditions could accelerate trehalose production within the worms to counteract the environmental change.

In conclusion, TPP serves as a critical regulator of energy metabolism and survival in T. spiralis. The specific inhibitor NPP disrupts glucose metabolic homeostasis in a dose-dependent manner, thereby suppressing TPP activity and compromising worm viability. These findings provide a theoretical foundation for the development of novel anti-T. spiralis agents that target TPP. Nevertheless, further studies are warranted to determine whether NPP impairs worm establishment and parasitism by interfering with larval migration and molting, to elucidate its precise mechanisms of action, and to evaluate its safety in subsequent animal experiments.

Acknowledgements

We thanks to Northeast Agricultural University and the National Parasitic Resources Center for their help in this study.

Author contributions

Ting Zhang and Rulin Ruan: Conceptualization, Methodology, Formal analysis and investigation. Jiaming Hou and Yongqing Yang: Formal analysis and investigation, Writing - Original Draft. Han Xue: Writing - Review & Editing. Mingxin Song and Caixia Han: Conceptualization, Supervision, Funding acquisition, Project administration, Writing - Review & Editing. All authors have read and approved the final manuscript.

Funding

This study was supported by the National Parasitic Resources Center (NPRC-2019-194-30).

Data availability

The data supporting the findings of this study are available within the article and its supplementary materials.The datasets used or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The feeding and experimental procedures of mice were carried out in accordance with the Chinese Animal Management Ordinance. The animal experiment standards were approved by the Animal Management Committee of Northeast Agricultural University, and implemented in accordance with animal ethics guidelines and approved protocols (Animal Ethics Committee approval number NEAUEC20210334).

Consent to publish

The manuscript has been approved by all authors for publication.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Jiaming Hou and Rulin Ruan contributed equally to this work.

Contributor Information

Mingxin Song, Email: songmx@neau.edu.cn.

Caixia Han, Email: hancaixia@neau.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data supporting the findings of this study are available within the article and its supplementary materials.The datasets used or analyzed during the current study are available from the corresponding author upon reasonable request.

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