Expression And Function Study On Thioredoxin Glutathione Reductase Of Schistosoma Japonicum

Schistosomiasis, caused by the blood-fluke from the genus Schistosoma, is a zoonotic parasitic disease which is harmful to human health. Currently, the disease is endemic in 12 provinces (prefectures, or antonomous regions) along the middle and lower reaches of the Yangtze River in China, with a further of more than 0.4 million people infected. The infection of the parasite can lead to parenchymal disease in humans, resulting in serious damages to human health. Schistosome has a complex life cycle, with continuous updating of surface antigen composition, and it can adjust the host immune system actively. And these make it has a strong capacity of immune evasion. As a consequence, it brings great difficulty for the development of vaccines against the disease. There are no schistosomiasis vaccines available around the world till now. Currently, the control of schistosomiasis mainly relies on drug-based chemotherapy.Since the praziquantel was developed in 1980s, a breakthrough during the period of the control of the disease, it has replaced other drugs and became the first and virtually the only choice of drug for the treatment of human schistosomiases. However, the characteristics of concomitant immunity of Schistosoma infection makes the host not be able to give rise to a long-lasting immunity against re-infection, and repeated treatment for the re-infections is essential. It implies that the parasites may develop resistance to the drug under the long-term, repeated large-scale chemotherapy. In 1994, the praziquantel-resistant strains in Schistosoma mansoni have been induced in laboratory by Doenhoff et al. Futhermore, decreased sensitivity of S. mansoni to praziquantel has been also found in some endemic areas of Africa and South America. There are also some cases reporting failure in treatment of S. haematobium infections using the routine treatment protocols. Artemisinin, which was previously acknowledged as antimalarial drugs, has also appeared activity on Schistosoma species. However, the drug suffers from the problem of being less sensitive to adult, and that the extensive use of artemisinin may develop drug resistance in malaria is also worried, and limits the further application of the drugs. Up to now, praziquantel is actually the only antischistosomicide available in schistosomiasis control program all over the world. Therefore, development of novel antischistosomal drugs instead of praziquantel is given high priority.In recent years, the redox balance of organisms has become an important target for development of new drugs gradually. In order to maintain the normal life activity, the parasitic pathogens in the host body must keep the cellular redox balance and must be able to evade attackment of the reactive oxygen species generated by the host's immune response. There are two major systems to detoxify reactive oxygen species in eukaryotes, one is thioredoxin system and another is glutathione system. In addition to protect against oxidative damage, these two systems also play an important role in regulation of transcriptional factor, cell growth and differentiation, synthesis of DNA, hormone action, cytokine function and metabolism of selenoprotein, etc. Currently, TrxR has become a very potential target for development of novel drugs against tumor and infectious pathogens.In helminth, like S. mansoni and Larval Taenia crassiceps (Cysticerci), it has been proved that there isn't separate TrxR and GR enzyme. The function of TrxR and GR is replaced by a unique multifunctional enzyme, thioredoxin glutathione reductase (TGR). The TGR of S. mansoni (SmTGR) has been confirmed as an essential gene for S. mansoni survival using RNA interference. Following the silencing of SmTGR expression or inhibition of the SmTGR activity, worms died of oxidative damage. SmTGR should be a promising and potential drug target against schistosomiasis. Up to now, there is no information pertaining to the thirodoxin glutathione reductase of Schistosoma japonicum (SjTGR) available now. In this study, the gene of SjTGR was cloned, the selenium protein of SjTGR was prepared, and the function characterization of this protein was investigated for the first time. The main content of the present thesis is listed as following.Part 1 Cloning and polymorphism investigation of SjTGR genemRNA was extracted from adult S. japonicum worms following the instruction of QuickPrepTM mRNA purification kit, and the first strand cDNA of S. japonicum worm was prepared according to the instruction of PhusionTM RT-PCR kit of Fermentas Life Sciences. The ESTs of SjTGR was found by searching the EST database of S. japonicum using the gene DNA sequence of SmTGR. A pair of gene specific primers was designed according to the sequence of homologous ESTs, and the DNA fragments of SjTGR gene was amplified from the worm cDNA, and cloned into pGEM-T vector to form the recombinant plasmids of SjTGR/pGEM-T. The sequencing data showed that SjTGR was a selenoprotein with the conserved sequence GCUG (U is Selenocysteine) at the carboxyl-terminal. The polymorphism of point mutation, deletion and others were present in the gene of SjTGR.Part 2 Construction of the chimeric gene expressing SjTGR selenium proteinIn order to prepare SjTGR with bioactivity, a bacterial-type SECIS element was fused to the end of stop codon UGA of the open reading frame of SjTGR gene following the reported strategy of selenoprotein expression to form a chimeric gene. This SECIS element could make the selenocysteine exactly insert into the position of UGA of SjTGR to form the selenoprotein during the translation, which made the SjTGR which was expressed in the E.coli has natural enzyme activity.Part 3 Expression and characterization of selenoprotein SjTGRA recombinant plasmid SjTGRsec/pET41a was constructed by subcloning the chimeric gene of SjTGR fused with bacterial-type SECIS element into expression plasmid pET41a. This plasmid was co-transfermed into host E. coli BL21 with plasmid pSU ABC. The soluble SjTGR protein was expressed successfully by inducing transformants with IPTG at static growth stage for 24 h at 24°C. The expressed products of plasmid pSU ABC could promote the integration of selenocysteine and increase the yield of selenoprotein testified by 75Se incorporation test. The recombinant SjTGR was purified from expression products by affinity chromatography with an adenosine 2', 5'- diphosphate agarose column. The polyclonal antiserum with high titer against recombinant SjTGR was obtained by immunizing mice with purified SjTGR protein. The result of Western Blotting showed that the native TGR existed in S. japonicum adult worms without separate TrxR and GR. Part 4 Enzyme kinetics of SjTGRThe activities of thiorendoxin reductase (TrxR),glutathione reductase (GR) and gluaredoxin (Grx) of recombinant TGR with the substrates 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), insulin, glutathione disulfide (GSSG), andβ-hydroxyethyl isulfide (HED) were analyzed at 25°C using the UV-visible spectrophotometer, and the Km value of each substrate was also calculated. The half maximal inhibitory concentration (IC50) and inhibitory constants (Ki) of auranofin on recombinant SjTGR were determined. The results showed the SjTGR was a multifunctional enzyme with activities of TrxR (8.11μmol·min-1mg-1), GR (2.19μmol·min-1mg-1) and Grx (12.10μmol·min-1mg-1). The Km of different substrates of TGR were 21.42±0.44μM (NADPH), 3.24±0.40μM (Trx), 145.05±6.31μM (DTNB), 49.55±6.31μM (GSSG), 2,792±231μM (HED), 3,831±54μM (GSH). Auranofin could inhibit the activity of SjTGR markedly. The IC50 values of auranofin against 5nM recombinant SjTGR were 6.89, 0.47, 8.12 nM with DTNB, GSSG, HED substrate respectively. The Ki values were 0.762 nM for TrxR activity and 0.034 nM for GR activity. The inhibition of auranofin on TrxR activity was competitive, while the inhibition of auranofin on GR activity was noncompetitive.Part 5 Toxicity of several compounds to S. japonicumThe drugs (auranofin, cisplatin, adriamycin,compounds 4N, H, B, O) with different concentrations were added into RPMI 1640 medium with Schistosoma japonicum adult worms, which had been cultured for 30-60 min. The activity, morphological changes and death of the worms were observed after 1 h, 6 h, 24 h, 48 h, 72 h respectively, then the worms were transferred to fresh medium without drugs to observe whether their activity would be recovered, and 50% lethal dose (LD50) of the drugs against adult worms was determined. The TrxR and GR activities of thioredoxin glutathione reductase of Schistosoma japonicum in homogenized supernatant of adult worms processed by drugs were tested. Each mouse was infected with 30 S. japonicum cercariae by abdominal skin penetration. The mice in different groups were administrated with 6 mg/kg auranofin and 45 mg/kg 4N intraperitoneally twice daily for 5 days at different developmental stages as following: skin-stage (started 1 d after infection); lung-stage (started 7 d after infection); liver-stage (started 23 d after infection); adult-stage (started 35 d after infection). Control mice were administrated with same dose of carrier (DMSO and sodium chloride) on the same timetable. Mice were sacrificed at 42th day post-infection, and any adult S. japonicum worms in the hepatic and portomesenteric veins were recovered and counted. The worm and egg reductions, which were caused obviously by the drug treatment, as percentages, were calculated. The result showed that 5μg/mL of auranofin for 24 h, 20μg/mL of 4N for 72 h, 60μg/mL of H for 72 h, and 80μg/mL of cisplatin for 72 h can damage 100%, 60%, 66.7% and 100% of adult worms, respectively, and there were statistically significant differences compared with the control group. The LD50 values for auranofin, 4N, H, and cisplatin were 2.56μg/mL, 17.59μg/mL, 54.14μg/mL, and 52.87μg/mL respectively, but no toxic effects of other drugs on schistosome worms were found. The toxic effects of auranofin, 4N, cisplatin, and H on adult worms were irreversible. Auranofin and cisplatin inhibited TGR activity of Schistosoma japonicum, but other drugs had no similar effect. 5-30μg/mL of auranofin, 20μg-30μg/mL of 4N, 70-150μg/mL of cisplatin, and 60-220μg/mL of H made the morphological changes of the worms 24h post-treatment. The in-vivo result showed that auranofin could result in 41.50% worm burden reductions and 43.18% egg burden reductions at adult worm stage, but had no effect on schistosomula (P values were all less than 0.05). 4N had no toxicity to any developmental stages of S. japonicum.Part 6 Screening and identification of inhibition peptides on SjTGR activity with phage display peptide libraryThe purified SjTGR was coated in petri dishes (60×15 mm) to react with 1011 pfu phage particles of phage display peptide library. The unbound phages were washed out with stringent conditions, and the phages binding to SjTGR specifically were eluted with NADPH and amplified. The amplified phage particles were used for the second screening with SjTGR coated in petri dishes. The panning screening was repeated for three times to obtain the specific phage clones binding to SjTGR. The DNA sequenced results showed that four homologous peptide were found in 58 phage clones sequenced after three rounds of panning screening, which named: JIPDys1, JIPDys2, JIPDys3, JIPDys4 respectively. JIPDys1 presented in 26 clones, JIPDys2 in 2 clones, JIPDys3 in 3 clones and JIPDys4 in 4 clones. The binding capacity of four piptides to SjTGR were confirmed further by ELISA and Western Blotting. Enzyme activity inhibition showed that JIPDys1, JIPDys2, JIPDys3 could inhibit the activity of SjTGR. The inhibiting effect of 1011 pfu phage JIPDys1, JIPDys2, JIPDys3 on TrxR, GR and Grx activity of TGR were 59.04%, 80.80%, 53.56%; 45.24%, 69.43%, 25.64%; 54.72%, 20.88%, 43.02% respectively. Peptide JIPDys4 did not inhibit the activity of SjRGR.ConclusionsIn the present study, SjTGR selenium protein with TrxR, GR and Grx activity was prepared successfully. It has been proved that SjTGR is a key molecule in maintaining the redox balance of worm and is an essential gene for Schistosoma japonicum survival by in vitro and In vivo test. SjTGR is a potential target for developing new drugs against schistosomiasis. The phage displayed peptides screened by phage display peptide library technique, could inhibit the activity of TGR, lays a good foundation for the further development of new drugs against schistosomiasis with independent intellectual property rights.