Effect of RNA interference with glutamate decarboxylase on acid resistance of Trichinella spiralis

Abstract

Trichinella spiralis is a zoonotic parasite that infects most mammals, even humans. Glutamate decarboxylase (GAD) is an important enzyme in glutamate-dependent acid resistance system 2 (AR2), but the GAD of T. spiralis in AR2 is unclear. We aimed to investigate the role of T. spiralis glutamate decarboxylase (TsGAD) in AR2. We silenced the TsGAD gene to evaluate the AR of T. spiralis muscle larvae (ML) in vivo and in vitro via siRNA. The results showed that recombinant TsGAD was recognized by anti-rTsGAD polyclonal antibody (57 kDa), and qPCR indicated that TsGAD transcription peaked at pH 2.5 for 1 h compared to that with pH 6.6 phosphate-buffered saline. Indirect immunofluorescence assays revealed that TsGAD was expressed in the epidermis of ML. After TsGAD silencing in vitro, TsGAD transcription and the survival rate of ML decreased by 15.2% and 17%, respectively, compared with those of the PBS group. Both TsGAD enzymatic activity and the acid adjustment of siRNA1-silenced ML were weakened. In vivo, each mouse was orally infected with 300 siRNA1-silenced ML. On days 7 and 42 post-infection, the reduction rates of adult worms and ML were 31.5% and 49.05%, respectively. Additionally, the reproductive capacity index and larvae per gram of ML were 62.51±7.32 and 1250.22±146.48, respectively, lower than those of the PBS group. Haematoxylin-eosin staining revealed many inflammatory cells infiltrating the nurse cells in the diaphragm of mice infected with siRNA1-silenced ML. The survival rate of the F1 generation ML was 27% higher than that of the F0 generation ML, but there was no difference from the PBS group. These results first indicated that GAD plays a crucial role in AR2 of T. spiralis. TsGAD gene silencing reduced the worm burden in mice, providing data for the comprehensive study of the AR system of T. spiralis and a new idea for the prevention of trichinosis.