Molecular characteristics of glutathione transferase gene family in a neglect medical Spirometra tapeworm

Abstract

The Spirometra mansoni is a neglect medical tapeworm, its plerocercoid larvae can parasitize in humans and animals, causing sparganosis. In this study, 17 new members of the glutathione transferase (GST) family were sequenced and characterized in S. mansoni. Clustering analysis displayed the categorization of SmGSTs into two main clades. RT-qPCR illustrated that 7 GST genes were highly expressed in the plerocercoid stage while 8 GSTs were highly expressed in the adult. rSmGST has the typical C- and N-terminal double domains of glutathione transferase. Immunolocalization revealed that natural SmGST is mainly located in the epidermis and parenchyma of plerocercoid, and in the epidermis, parenchyma, uterus and egg shell of adult worm. The optimum activity for rSmGST was found to be pH 6.5 and 25°C. The evolutionary tree showed a high level of diversity of cestodes GSTs. SmGSTs contained both conserved family members and members in the process of further diversification. The findings in this study will lay a foundation to better explore the underlying mechanisms of GSTs involved in Spirometra tapeworms.

Keywords: cestode; enzymatic traits; glutathione transferase; molecular characterization; phylogeny.

Figure 1

Glutathione transferase protein family members identified in Spirometra mansoni. (A) Phylogenetic tree and conserved motifs of SmGSTs. The symbol “▴” indicates microsomal GSTs (MAPEG), “•” indicates Mu class cGST, “°” indicates Sigma class cGST, “⊗” indicates unknown class cGST. (B) Model quality evaluation of 3D structure of SmGST. Red represents the most favored regions, brown indicates additional allowed regions, yellow indicates the generously allowed regions, light color indicates disallowed regions. (C) Ribbon protein structures of SmGST. Swiss-model protein modeling server was used for modeling; α-helices show as ribbons and β-strands as arrows. Pink: the thioredoxin-like domain (N-terminal GSH binding domain), Green: C-terminal substrate binding domain. (D) GST genes expression of S. mansoni in different tissues by qRT-PCR. The expression level was normalized to GAPDH and measured with 2^−ΔΔCt value. Results are averaged from three independent replicates during all stages. Error bars represent SD (n = 3). IMP, immature proglottide; MP, mature proglottide; GP, gravid proglottide.

Figure 2

Molecular characterization of cloned SmGST. (A) Determination of optimal antigen coating concentration. (B) Determination of anti-rSmGST immune serum titer by indirect ELISA. Red, blue, green, purple, and orange represent serum dilutions of 1:10², 1:10³, 1:10⁴, 1:10⁵, and 1:10⁶, respectively. (C) SDS-PAGE analysis of 10 μg purified GST from S. mansoni on 12% gel. M: protein pre-staining marker; Lane 1: uninduced bacterial cultures; Lane 2: the lysate of the induced recombinant bacteria harboring pQE-80L-rSmGST after ultrasonication; Lane 3: rSmGST purified by Ni-NTA-Sefinose Column. (D) rSmGST antigenicity analysis. M: protein pre-staining marker; Lane 1: rSmGST + anti-rSmGST serum; Lane 2: rSmGST + infected mouse serum; Lane 3: rSmGST + pre-immune serum. (E) The transcription pattern of GST gene in different developmental stages of Spirometra mansoni. Conventional RT-PCR (upper) and real-time RT-PCR (lower) were performed on cDNA from various developmental stages of S. mansoni, including eggs, plerocercoid and adult. A house keeping gene (Se-GAPDH) was used as a positive control. H₂O was used as a negative control.

Figure 3

Immunofluorescence localization of GST in different developmental stages of Spirometra mansoni. (A) GST location in different segments in the adult stage. IMPR indicates immature proglottide, NESG indicates neck segment. (B) GST location in the plerocercoid stage. Green fluorescence is the location of GST protein. Scale of different segments of adult: 500 μm; Head of plerocercoid: 200 μm; Body of plerocercoid: 500 μm.

Figure 4

Enzymatic characteristics of SmGST. (A) Effects of temperature on rSmGST enzymatic activity. (B) Effects of pH on rSmGST enzymatic activity. (C) Effects of substrate concentration of GSH on rSmGST enzymatic activity. The kinetic parameters, K_m and V_max, were determined using the Lineweaver–Burk's plot. The K_m and V_max values were 0.78 mM and 1.146 μmol min⁻¹ mL⁻¹, respectively. (D) Effects of substrate concentration of CDNB on rSmGST enzymatic activity. The K_m and V_max values were 6.29 mM and 10.120 μmol min⁻¹ mL⁻¹, respectively. (E) The effect of different concentrations of Rose Bengal (RB) on the initial velocities. (F) The effect of different concentrations of Tripheniltin chloride (TPT) on the initial velocities. (G) The effect of different concentrations of Bromosulfophtalein (BSP) on the initial velocities. (H) The effect of different concentrations of Cibacron Blue (CB) on the initial velocities. Inset shows secondary plot of the 1/V_max values derived from the primary Lineweaver–Burk plot vs. concentration for the determination of K_i. (E) RB (20, 60 μM); (F) TPT (4, 8 μM); (G) BSP (20, 60 μM); (H) CB (10, 30 μM).

Figure 5

Conserved motifs of GST sequence motifs in medical cestodes.

Figure 6

Phylogenetic analysis of glutathione transferase sequences in selected medical cestodes based on maximum likelihood method. The numbers on the branches represent bootstrap values, and only values with bootstrap values >60 are displayed.