Molecular expression and enzymatic characterization of thioredoxin 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 years. Defense against oxidative damage can be mediated through glutathione and/or thioredoxin utilizing systems. Here, we report the molecular expression and biochemical characterization of a thioredoxin (Trx) from O. viverrini. O. viverrini Trx cDNA encoded a polypeptide of 105 amino acid residues, of molecular mass 11.63 kDa. The predicted protein has similarity to previously characterized thioredoxins with 26-51% identity. Recombinant O. viverrini Trx (Ov-Trx-1) was expressed as soluble protein in E. coli. The recombinant protein showed insulin reduction activity and supported the enzymatic function of O. viverrini thioredoxin peroxidase. Expression of Ov-Trx-1 at mRNA and protein levels was observed in all obtainable developmental stages of the liver fluke. Ov-Trx-1 was also detected in excretory-secretory products released by adult O. viverrini. Immunohistochemistry, Ov-Trx-1 was expressed in nearly all parasite tissue excepted ovary and mature sperms. Interestingly, Ov-Trx-1 was observed in the infected biliary epithelium but not in normal bile ducts. These results suggest that Ov-Trx-1 is essential for the parasite throughout the life cycle. In the host-parasite interaction aspect, Ov-Trx-1 may support thioredoxin peroxidase in protecting the parasite against damage induced by reactive oxygen species from inflammation.

Fig. 1

Multiple alignment of deduced amino acid sequences of O. viverrini thioredoxin with thioredoxin from other species: S. mansoni (AAL79841), F. hepatica (AAF14217), E. granulosus (AAC14584), H. sapiens (NP_003320), M. musculus (NP_035790) and E. coli (AAA24696). The conserved active site WCGPC residues are boxed and two cysteines of active site are indicated by arrow head.

Fig. 2

Phylogenetic tree of Trx family members, including Ov-Trx-1 from O. viverrini (EL620399) (in the box); flatworm, F. hepatica (AAF14217); roundworm, Ascaris suum (AAS78778), Brugia malayi (XP_001900802); plant, Eucalyptus grandis (ABB53600), Medicago truncatula (AAZ98843), Brassica juncea (ABM54166); archeae, Sulfolobus solfataricus (NP_343612), Archaeoglobus fulgidus (NP_070969); bacteria, E. coli (AAA24696), Clostridium perfringens (NP_563271); fungi, Aspergillus fumigatus (XP_751916), Pichia stipitis (XP_001387891); and vertebrates, H. sapiens (NP_003320), Bos Taurus (NP_776393), M. musculus (NP_035790). 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-Trx-1: Proteins were separated under SDS-PAGE; lane 1, E. coli lysate; lane 2, E. coli lysate after affinity purification with Ni-NTA affinity chromatography and lane3, purified Ov-Trx-1.

Fig. 4

Insulin reduction by recombinant Ov-Trx-1. Reactions with 1.5μM, 0.3μM and without (0μM) of recombinant Ov-Trx-1 are indicated. All reactions contain 0.33mM DTT.

Fig. 5

Hydrogen peroxide reduction by recombinant Ov-TPx-1 supported by recombinant Ov-Trx-1. The rate of hydrogen peroxide consumption (μmol/min) by recombinant recombinant Ov-TPx-1 in the presence of variable amounts of recombinant Ov-Trx-1 (in μM).

Fig. 6

Expression profile of Ov-Trx-1 in different developmental stages of O. viverrini by conventional RT-PCR: lane 1, egg; lane 2, metacercaria; lane 3, 2 weeks juvenile worm; lane 4, 1 month adult worm and lane 5, 2 months adult worm. Ov-β-actin and Ov-TPx-1 were used as control.

Fig. 7

Western blot analysis of Ov-Trx-1 in different developmental stages of O. viverrini: lane 1, recombinant Ov-Trx-1; lane 2, egg extract; lane 3, metacercaria extract; lane 4, adult worm extract; lane 5, excretory-secretory products.

Fig. 8

Immunohistochemistry of Ov-Trx-1 in adult O. viverrini and infected bile ducts. The thioredoxin is moderately to highly expressed in tegumental cells (T, arrow) and parenchyma, gut epithelium (G), and eggs (E) in the uterus but not ovary (O) (B). The infected biliary epithelium that lining close to the fluke shows intense thioredoxin staining. Negative control using preimmune mouse serum exhibits no staining at all (A). (Immunoperoxidase staining, original magnification 100X).