Fig 1.
Multi sequence alignment of pyruvate kinase of different species/genes of Trichinella.
According to the analysis of Cluster Omega, the same amino acids are marked in blue and conservative substitution of amino acid residues are marked in light blue. The pyruvate kinase genes of different species/genotypes of Trichinella have a high homology. The number at the end of each sequence represents the percentage of identity with TsPKM.
Fig 2.
Tertiary structure prediction of TsPKM and evolutionary tree construction of pyruvate kinase of 13 organisms.
A: Tertiary structure prediction of TsPKM. Eight amino-acid residues (Arg, Asn, Asp, Phe, Lys, Glu, Asp and Thr) constitute the enzyme active site signed as purple. B: TsPKM in the evolutionary tree of Trichinella, human and mouse. The evolutionary tree of pyruvate kinase of 11 different species/genotypes of the genus Trichinella was constructed by neighbor-joining (NJ) method. The encapsulated and non-encapsulated Trichinella was localized in two different evolutionary clades of Trichinella.
Fig 3.
Expression and identification of rTsPKM.
A: SDS-PAGE analysis of the rTsPKM. Lane M: Protein marker; Lane 1: lysate of recombinant E. coli incorporating pQE-80L/TsPKM prior to induction; Lane 2: lysate of recombinant E. coli incorporating pQE-80L/TsPKM after induction; Lane 3: purified rTsPKM. B. Western blotting analysis of rTsPKM antigenicity. Lane M: Protein marker; Lane 1: lysates of pQE-80L/TsPKM prior to induction were not recognized by infection serum. Lane 2: lysates of pQE-80L/TsPKM after induction were recognized by infection serum. The purified rTsPKM was recognized by anti-rTsPKM serum (lane 3), infection serum (lane 4) and anti-His tag McAb (lane 6), but not by normal serum (lane 5).
Fig 4.
Transcription and expression of TsPKM in different stages of Trichinella spiralis.
A: RT-PCR analysis of TsPKM transcription in diverse stages. Lane M: DNA marker; Lane 1: ML. Lane 2: 6 h IIL. Lane 3: 3 d AW. Lane 4: NBL. B: SDS-PAGE analysis of crude proteins of diverse worm stages. Lane M: protein marker. Lane 1: ML soluble protein. Lane 2: IIL soluble protein. Lane 3: 3 d AW soluble protein. Lane 4: NBL soluble protein. C: Western blot analysis of crude proteins of diverse worm stages of ML (lane 1), IIL (lane 2), 3 d AW (lane 3) and NBL (lane 4) identified using anti-rTsPKM serum. D: SDS-PAGE analysis of ES proteins of ML (lane 1), IIL (lane 2) and 6 d AW (lane 3), Lane M: protein marker. E: Western blot analysis of ES proteins of ML (lane 1), IIL (lane 2) and 6 d AW (lane 3) recognized by anti-rTsPKM serum. The recognized native TsPKM with about 58.5 kDa were indicated with arrows.
Fig 5.
Expression of TsPKM at the cuticle of various T. spiralis stages by IFT.
The whole intact worms were probed by anti-rTsPKM serum, and immune fluorescence staining was observed at the epicuticle of ML, IIL, NBL and the intestine of 3 d AW. But pre-immune normal serum did not recognize any worm components of the nematode. Scale bars: 100 μm.
Fig 6.
Immunolocalization of TsPKM in worm cross-sections of diverse T. spiralis stages by IFT with anti-rTsPKM serum.
Green fluorescence staining was observed at cuticle, muscle, midgut, stichosome, and female intrauterine embryos. No immunostaining in worm cross-sections was observed by normal serum as a negative control. Scale-bars: 100 μm.
Fig 7.
Enzyme activity analysis of rTsPKM.
rTsPKM was incubated with 2.5 mm PEP and 1.25mm ADP for 10 min under various conditions. The optimal catalytic conditions of rTsPKM were assessed with various rTsPKM concentrations (1–12 ng/μl), temperatures (20–70°C) and buffer solution with different pH (4–10). A: The optimum catalytic concentration of rTsPKM is 10 ng/μl. B: The optimum catalytic temperature of rTsPKM is 37°C. C: The optimum catalytic pH of rTsPKM is 8.0. D: Effects of different metal ions on rTsPKM activity. K+ and Mg2+ have obvious enhancement role on rTsPKM activity. E: Effects of different inhibitors on rTsPKM activity. F and G: The suppressive role of tannin (F) and ethyl pyruvate (G) on rTsPKM activity is dose-dependent. H: Standard curve of sodium pyruvate. I: Michaelis–Menten curve and Lineweaver–Burk of PEP at pH 8.0 and 37°C. J: Michaelis–Menten curve and Lineweaver–Burk of ADP at pH 8.0 and 37°C.
Fig 8.
Suppression of tannin (A) and ethyl pyruvate (B) on native TsPKM enzymatic activity in T. spiralis muscle larval somatic proteins. *P < 0.0001 relative to the saline control group.
Fig 9.
Silencing TsPKM gene suppressed TsPKM expression and enzymatic activity.
A: TsPKM transcription levels in ML transfected with different dsRNA-TsPKM. B: TsPKM expression levels in ML transfected with different dsRNA-TsPKM. C: TsPKM transcription levels in ML transfected with various doses of dsRNA-TsPKM2. D: TsPKM expression levels in ML transfected with various doses of dsRNA-TsPKM2. E: TsPKM transcription levels in ML at 1–3 days after transfection with 60 ng/μl dsRNA-TsPKM2. F: TsPKM expression levels in ML at 1–3 days after transfection with 60 ng/μl dsRNA-TsPKM2. G: Expression levels of TsPKM and Trichinella spiralis calreticulin (TsCRT) in ML treated using dsRNA-TsPKM2. H: TsPKM enzyme activity in dsRNA-TsPKM2 treated in ML. *P < 0.05 relative to the PBS group.
Fig 10.
Suppression of dsRNA-TsPKM (A) and tannin (B) on larval ATP content.
*P < 0.05 relative to the PBS or saline control group.
Fig 11.
Suppression of dsRNA-TsPKM on T. spiralis larval glycometabolism.
Glycogen is mainly distributed in the muscle larval stichosome (A) and around the intestine (B). C: dsRNA-TsPKM reduced larval total sugar content. D: Tannin reduced larval total sugar content. The arrows indicate glycogen. *P < 0.0001 relative to the PBS or saline group. Scale bars: 100 μm.
Fig 12.
Suppression of dsRNA-TsPKM and tannin on T.
spiralis larval lipid metabolism A: Distribution of lipid droplets in different groups of T. spiralis muscle larvae. Small lipid droplets were distributed all over the muscle larvae, but large lipid droplets were principally localized in the larval intestine and tail. After treatment with dsRNA-TsPKM and tannin, the larval red color became lighter, indicating that larval lipid content of treated larvae with dsRNA-TsPKM and tannin was obviously lower than the control groups. B: dsRNA-TsPKM decreased larval lipid content, C: tannin decreased larval lipid content. *P < 0.0001 relative to the PBS or saline group. Scale bars: 100 μm.
Fig 13.
Trichinella spiralis larval molting was suppressed by dsRNA-TsPKM in the IIL stage.
A: RNAi and tannin significantly inhibited the larval molting, and there was no transparent sheath on both the anterior and tail ends. Especially in the tannin group, there was no obvious separation between old and new cuticles. The area in the red box was enlarged for observation. B: Molting of T. spiralis larvae at 3 days after RNAi and tannin treatment. *P < 0.05 relative to the PBS group. #P < 0.01 relative to the saline group. Scale bars: 200 μm.
Fig 14.
RNAi suppressed the in vivo larval development.
The numbers of 24 h IIL (A) and 3 d AW (B) collected from intestine of mice infected with the dsRNA-TsPKM and tannin treated ML. The length of 24 h IIL (C) and 3 d female (D) and male adult worms (E) collected from various groups of infected mice. *P < 0.001 relative to the PBS or saline control group.
Fig 15.
Inhibition of specific dsRNA-TsPKM on native TsPKM enzymatic activity of 24 h IIL (A) and 3 d AW (B) collected from mice challenged with T. spiralis ML treated by dsRNA-TsPKM and tannin. *P < 0.0001 relative to the PBS or saline control group.
Fig 16.
Suppression of dsRNA-TsPKM on sugar and lipid metabolism of T. spiralis adult worm from infected mice.
dsRNA-TsPKM evidently reduced the content of sugar (A) and lipid (B) in 3 d adult worms. *P < 0.0001 relative to the PBS or saline group.