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.

Fig 1

A: Analysis and multi-alignment of rTsp-GILT sequence with GILT of other known species. Identical amino acids among all sequences are indicated by black background. The active-site motif of CXXC and GILT signature sequences are highlighted in red boxes. The predicted signal peptide is marked with green underlining. The glycosylation sites are indicated by blue underlining. The conserved cysteines sites were marked by black ◇. B: Alignment of the predicted 3D structure of T. spiralis with H. sapiens and S. scrofa. C: The phylogenetic tree of 13 GILT proteins.

Fig 2

A: Purification of Recombinant protein. M: Protein molecular weight Marker; Lane 1: Supernatant of recombinant E. coli lysis; Lane 2: Inclusion body from recombinant E. coli; Lane 3: Purified recombinant protein. B: Tsp-GILT exhibits thiol reductase activity in vitro. M, protein molecular weight marker; Lane 1: Rabbit IgG treated without DTT at pH 7.0 as negative control; Lane 2: Rabbit IgG treated with 50 mM DTT at pH 7.0 as positive control; lane 3:Rabbit IgG incubated with Tsp-GILT (activated by 10 mM DTT at pH 4.5); Lane 4:Rabbit IgG incubated with Tsp-GILT (without DTT at pH 4.5); Lane 5: Rabbit IgG incubated without Tsp-GILT, with 10 mM DTT at pH 4.5; Lane 6: Rabbit IgG incubated with pET32a tag protein (treated with 10 mM DTT at pH 4.5); Lane 7: Rabbit IgG incubated with Tsp-GILT (treated with 10 mM DTT at pH 7.0).

Fig 3. Localization of Tsp-GILT in T. spiralis ML by immunofluorescence assay.

First line is negative control group, second line is experience group. Nuclei were stained with DAPI (blue) and target protein with Cy3 (red). No red fluorescence was observed in control.

Fig 4

Transcription level of Tsp-GILT by QRT-PCR during various development stages of T. spiralis. NBL: Newborn Larva (relative fold = 1.80); ML: Muscle Larva(relative fold = 1); AD; Adult worms(relative fold = 0.58). Significant differences were observed as *P < 0.05.

Fig 5. QRT-PCR analysis of Tsp-GILT expression during muscle larval stage treated with different concentration of IFN-γ and PBS (control).

The significant level was set at *P < 0.05, and ns non-significant compared with control group.

Fig 6. Knockdown of Tsp-GILT gene in ML and NBL.

A: Tsp-GILT mRNA transcription level of MLs treated with siRNA1, siRNA2, siRNA3, control siRNA and PBS. B: Tsp-GILT mRNA transcription level of NBLs treated with siRNA2, control siRNA and PBS. C: Immunofluorescence assay of siRNA treated ML and NBL. Row 1: PBS treated ML; Row 2; Cy3 labeled control siRNA treated ML. Row 3: PBS treated NBL; Row 4; Cy3 labeled control siRNA treated NBL. Control siRNA treated ML and NBL showed transformation indicating red color. While, no color was observed in both control groups. D: Survival rate (%) of ML and NBL (E) in vitro after suppression of Tsp-GILT expression by siRNA2. The significant level was set at *P < 0.05, and ns non-significant compared with control group.

Fig 7. Infectivity of siRNA2 treated ML.

A: Number of adult worms. B: Number of NBLs. C: Number of next generation MLs. The significant level was set at **P < 0.01, ***P < 0.001, and ns non-significant compared with control group.

Fig 8. Infectivity of siRNA2 treated NBL.

Number of ML population developed from siRNA2 treated NBLs. The significant level was set at **P < 0.01, ***P < 0.001, and ns non-significant compared with control group.