Characterization of the antioxidant enzyme, thioredoxin peroxidase, from the carcinogenic human liver fluke, Opisthorchis viverrini

Abstract

The human liver fluke, Opisthorchis viverrini, induces inflammation of the hepatobiliary system. Despite being constantly exposed to inimical oxygen radicals released from inflammatory cells, the parasite survives for many years. The mechanisms by which it avoids oxidative damage are unknown. In this study, thioredoxin peroxidase (TPx), a member of the peroxiredoxin superfamily, was cloned from an O. viverrini cDNA library. O. viverrini TPx cDNA encoded a polypeptide of 212 amino acid residues, of molecular mass 23.57kDa. The putative amino acid sequence shared 60-70% identity with TPXs from other helminths and from mammals, and phylogenetic analysis revealed a close relationship between TPxs from O. viverrini and other trematodes. Recombinant O. viverrini TPx was expressed as soluble protein in Escherichia coli. The recombinant protein dimerized, and its antioxidant activity was deduced by observing protection of nicking of supercoiled plasmid DNA by hydroxyl radicals. Antiserum raised against O. viverrini TPx recognized native proteins from egg, metacercaria and adult developmental stages of the liver fluke and excretory-secretory products released by adult O. viverrini. Immunolocalization studies revealed ubiquitous expression of TPx in O. viverrini organs and tissues. TPx was also detected in bile fluid and bile duct epithelial cells surrounding the flukes 2 weeks after infection of hamsters with O. viverrini. In addition, TPx was observed in the secondary (small) bile ducts where flukes cannot reach due to their large size. These results suggested that O. viverrini TPx plays a significant role in protecting the parasite against damage induced by reactive oxygen species from inflammation.

Fig. 1

Multiple alignment of deduced amino acid sequences of Opisthorchis viverrini TPx with 2-Cys Prx from other species: Schistosoma mansoni (TPx, AAD40685), Schistosoma japonicum (TPx, BAD90102), Fasciola hepatica (TPx, P91883), Caenorhabditis elegans (Prx2, AAN63412), Taenia solium (Prx, AAV91322) and Homo sapiens (Prx1, NP_002565). Two conserved domains of 2-Cys Prx (FYPLDFTFVCPTELIA and VCPA) are boxed and VCP motifs are indicated by letters above the sequences. Predicted signal peptide of Ov-TPx-1 has been removed from the sequence in the alignment.

Fig. 2

Phylogenetic tree of TPx family members, including Ov-TPx-1 from Opisthorchis viverrini (in the box); archeae, Thermoplasma volcanium (TPx, BAB59365); bacteria, Prochlorococcus marinus (TPx, CAE20929), Leptospira interrogans (TPx, AAN50008), Helicobacter pylori (TPx, AAV33156), Rickettsia typhi (TPx, AAU03798); fungi, Candida albicans (TPx, XP_716082), Phanerochaete chrysosporium (Prx, AAV53576); flatworm, Echinococcus granulosus (TPx, AAD02002), Taenia solium (Prx, AAV91322), Fasciola hepatica (TPx, P91883), Schistosoma japonicum (TPx, BAD90102), Schistosoma mansoni (TPx, AAD40685); roundworm, Caenorhabditis elegans (Prx2, AAN63412), Dirofilaria immitis (TPx, AAC38831), Onchocerca volvulus (TPx, AAC48312); and vertebrates, Homo sapiens (Prx1, NP_002565), Canis familiaris (Prx4, XP_859371), Mus musculus (Prx4, NP_058044), Bos taurus (Prx4, AAG53660). The number on each branch represents the bootstrap value from 1000 replicates. Bootstrap values under 50% are omitted.

Fig. 3

Expression and purification of recombinant Ov-TPx-1: Proteins were separated under native polyacrylamide gel electrophoresis; lane 1, molecular weight markers; lanes 2, 3 and 4, E. coli lysate, E. coli lysate after affinity purification with TALON® resin, and, purified Ov-TPx-1 treated with denaturing sample buffer, respectively; lane 5, purified Ov-TPx-1 treated with non-denaturing, non-reducing sample buffer.

Fig. 4

Antioxidant activity of Ov-TPx-1. Lane 1, pUC19 plasmid; lane 2, pUC19 plasmid + FeCl₃; lane 3, pUC19 plasmid + FeCl₃ + DTT; lanes 4-9, pUC19 plasmid + FeCl₃ + DTT + recombinant Ov-TPx-1 (200, 100, 5,0 25, 12.5, 6.125 μg/ml, respectively); lane 10, pUC19 plasmid + FeCl₃ + DTT + 200 μg/ml recombinant O. viverrini thioredoxin. Supercoiled (SF) and nicked (NF) forms of pUC19 plasmid are indicated on the right.

Fig. 5

Western blot analysis of Ov-TPx-1 in different developmental stages of O. viverrini: lane 1, recombinant Ov-TPx-1 stored at 4°C for 1 day; lane 2, recombinant Ov-TPx-1 stored at -80°C for one year); lane 3, egg extract; lane 4, metacercaria extract; lane 5, adult worm extract; lane 6, excretory-secretory products.

Fig. 6

Immunolocalization of Ov-TPx-1 in adult O. viverrini and in infected hamster livers. Ov-TPx-1 is observed in all tissues of the fluke including stromal parenchyma, vitelline glands, testis, ovary, eggs, miracidium, gut epithelium. During early infection (1 week), Ov-TPx-1 is observed mainly in the worm but not in the biliary epithelium (A). From 2 weeks post-infection, Ov-TPx-1is also seen in the biliary epithelium, particularly that lying in close contact with the flukes (B-E). Ov-TPx-1 is also detected in small bile duct epithelia where the fluke cannot inhabit (F). A = 1 week, B = 2 weeks, C = 3 weeks, D = 4 weeks, E-F = 6 months post-infection. Immunoperoxidase staining, original magnification, 200 X (A-D, F) and 100X (E)