Trichinella spiralis: Knockdown of gamma interferon inducible lysosomal thiol reductase (GILT) results in the reduction of worm burden

Abstract

Trichinella spiralis is mammalian skeletal muscles parasite which may cause trichinellosis in animals and humans. Gamma interferon inducible lysosomal thiol reductase (GILT) is a widespread superfamily which plays key role in processing and presentation of MHC class II restricted antigen by catalyzing disulfide bond reduction. There are no reports about GILT in T. spiralis. In present study, GILT from T. spiralis (Tsp-GILT) was cloned, analyzed by multiple-sequence alignment, and predicted by 3D structure model. Recombinant Tsp-GILT (about 46 kDa) was efficiently expressed in Escherichia coli and thiol reductase activity suggested that in acidic environment the addition of a reducing agent is needed. Soaking method was used to knockdown expression of Tsp-GILT using small interference RNA (siRNA). Immunofluorescence assay confirmed the transformation of siRNA into muscle larva (ML) and new born larva (NBL). Quantitative real time-PCR (QRT-PCR) analysis revealed that transcription level of Tsp-GILT mRNA can be up-regulated by stimulation of mouse IFN-γ and down-regulated by siRNA2 in vitro. NBLs soaked with siRNA2 showed 32.3% reduction in the generation of MLs. MLs soaked with siRNA2 showed 26.2% reduction in the next generation of MLs, but no significant effect was observed on adult worms or NBLs. These findings concluded that GILT may play important roles in the development of T. spiralis parasite.

Inhibition of Trichinella spiralis Membrane-Associated Progesterone Receptor (MAPR) Results in a Reduction in Worm Burden

“…Simultaneously, siRNA-559 significantly inhibits the Ts-MAPRC2 protein more effectively than siRNA-419 ( p ≤ 0.05). This study supports the hypothesis that siRNA-Ts-MAPRC2 effects on worm reproduction might be mediated by specific steroid-binding domains in Ts-MAPRC2 [ 24 , 36 , 43 ]. In subsequent experiments, mRNA levels in ML were determined for all the treated groups.…”

“…Based on the results ( Figure 6 ), the number of muscle larvae collected (MLs) was significantly lower at 1:50 (rTs-MAPRC2-Ab ratio) (p ≤ 0.05). Several other studies also support these results, including Fei et al, Gagliardo et al, and Cui et al [ 35 , 36 , 49 ].…”

“…The number of NBL was also assessed and collected by culturing 50 adult female worms per group in RPMI-1640 with FBS (20%), penicillin (500 units/mL), and streptomycin (500 mg/mL) for 24 h at 37 °C with 5% CO 2 . MLs were collected from all five remaining mice in each group by digestion after euthanasia at 35 dpi [ 36 ].…”

“…The mice of every group were euthanized at 35 dpi, and the MLs were collected by artificial digestion as described above. Based on treatment with rTs-MAPRC2-Ab (1:50, 1:200, and 1:800 dilutions), and in comparison, to controls pET32a rat serum (control group) and PBS (control group), ML reductions were calculated [ 36 ].…”

“…At 35 dpi, the mice of each group were euthanized, and the MLs were collected by artificial digestion as described above. The reductions in MLs were calculated based on treatment with siRNAs (siRNA-180, siRNA-419, siRNA-559) compared to siRNA-Control and the PBS control [ 36 ].…”