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

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.