BackgroundClonorchiasis, caused by the infection of Clonorchis sinensis (C. sinensis), is a kind of neglected tropical disease, but it is highly related to cholangiocarcinoma and hepatocellular carcinoma (HCC). It has been well known that the excretory/secretory products of C. sinensis (CsESPs) play key roles in clonorchiasis associated carcinoma. From genome and transcriptome of C. sinensis, we identified one component of CsESPs, severin (Csseverin), which had three putative gelsolin domains. Its homologues are supposed to play a vital role in apoptosis resistance of tumour cell.Methodology/Principal FindingsThere was significant similarity in tertiary structures between human gelsolin and Csseverin by bioinformatics analysis. We identified that Csseverin expressed at life stage of adult worm, metacercaria and egg by the method of quantitative real-time PCR and western blotting. Csseverin distributed in vitellarium and intrauterine eggs of adult worm and tegument of metacercaria by immunofluorence assay. We obtained recombinant Csseverin (rCsseverin) and confirmed that rCsseverin could bind with calciumion in circular dichroism spectrum analysis. It was demonstrated that rCsseverin was of the capability of actin binding by gel overlay assay and immunocytochemistry. Both Annexin V/PI assay and mitochondrial membrane potential assay of human hepatocarcinoma cell line PLC showed apoptosis resistance after incubation with different concentrations of rCsseverin. Morphological analysis, apoptosis-associated changes of mitochondrial membrane potential and Annexin V/PI apoptosis assay showed that co-incubation of PLC cells with rCsseverin in vitro led to an inhibition of apoptosis induced by serum-starved for 24 h.Conclusions/SignificanceCollectively, the molecular properties of Csseverin, a molecule of CsESPs, were characterized in our study. rCsseverin could cause obvious apoptotic inhibition in human HCC cell line. Csseverin might exacerbate the process of HCC patients combined with C. sinensis infection.

Clonorchis sinensis has been classified as group I biological carcinogen for cholangiocarcinoma by the World Health Organization. Biological studies on excretory/secretory products (ESPs) enabled us to understand the pathogenesis mechanism of C. sinensis and develop new strategies for the prevention of clonorchiasis. In this study, sequence analysis showed that annexin B30 from C. sinensis (CsANXB30) is composed of four annexin repeats which were characterized by type II and III Ca(2+)-binding sites or KGD motif with the capability of Ca(2+)-binding. In addition, immunoblot assay revealed that recombinant CsANXB30 (rCsANXB30) could be recognized by the sera from rats infected with C. sinensis and the sera from rats immunized by CsESPs. Real-time PCR showed that its transcriptional level was the highest at the stage of metacercaria. Immunofluorescence assay was employed to confirm that CsANXB30 was distributed in the tegument, intestine, and egg of adult worms, as well as the tegument and vitellarium of metacercaria. rCsANXB30 was able to bind phospholipid in a Ca(2+)-dependent manner and human plasminogen in a dose-dependent manner. Moreover, cytokine and antibody measurements indicated that rats subcutaneously immunized with rCsANXB30 developed a strong IL-10 production in spleen cells and a high level of IgG1 isotype, indicating that rCsANXB30 could trigger specific humoral and cellular immune response in rats. The present results implied that CsANXB30 might be involved in a host-parasite interaction and affected the immune response of the host during C. sinensis infection.

Clonorchiasis, caused by direct and continuous contact with Clonorchis sinensis, is associated with hepatobiliary damage, inflammation, periductal fibrosis, and the development of cholangiocarcinoma. Hepatic stellate cells respond to liver injury through production of proinflammatory mediators which drive fibrogenesis; however, their endogenous sources and pathophysiological roles in host cells were not determined. C. sinensis ferritin heavy chain (CsFHC) was previously confirmed as a component of excretory/secretory products and exhibited a number of extrahepatic immunomodulatory properties in various diseases. In this study, we investigated the expression pattern and biological role of CsFHC in C. sinensis. CsFHC was expressed throughout life stages of C. sinensis. More importantly, we found that treatment of human hepatic stellate cell line LX-2 with CsFHC triggered the production of free radicals via time-dependent activation of NADPH oxidase, xanthine oxidase, and inducible nitric oxide synthase. The increase in free radicals substantially promoted the degradation of cytosolic IκBα and nuclear translocation of NF-κB subunits (p65 and p50). CsFHC-induced NF-κB activation was markedly attenuated by preincubation with specific inhibitors of corresponding free radical-producing enzyme or the antioxidant. In addition, CsFHC induced an increased expression level of proinflammatory cytokines, IL-1β and IL-6, in NF-κB-dependent manner. Our results indicate that CsFHC-triggered free radical-mediated NF-κB signaling is an important factor in the chronic inflammation caused by C. sinensis infection.

No abstract

BackgroundClonorchiasis is a globally important, neglected food-borne disease caused by Clonorchis sinensis (C. sinensis), and it is highly related to cholangiocarcinoma and hepatocellular carcinoma. Increased molecular evidence has strongly suggested that the adult worm of C. sinensis continuously releases excretory-secretory proteins (ESPs), which play important roles in the parasite-host interactions, to establish successful infection and ensure its own survival. Myoglobin, a hemoprotein, is present in high concentrations in trematodes and ESPs. To further understand the biological function of CsMb and its putative roles in the interactions of C. sinensis with its host, we explored the molecular characterization of CsMb in this paper.MethodsWe expressed CsMb and its mutants in E. coli BL21 and identified its molecular characteristics using bioinformatics analysis and experimental approaches. Reverse transcription PCR analysis was used to measure myoglobin transcripts of C. sinensis with different culture conditions. The peroxidase activity of CsMb was confirmed by spectrophotometry. We co-cultured RAW264.7 cells with recombinant CsMb (rCsMb), and we then measured the production of hydrogen peroxide (H2O2) and nitric oxide (NO) in addition to the mRNA levels of inducible nitric oxide synthase (iNOS), Cu-Zn superoxide dismutase (SOD1) and Mn superoxide dismutase (SOD2) in activated RAW264.7 cells.ResultsIn the in vitro culture of adult worms, the transcripts of CsMb increased with the increase of oxygen content. Oxidative stress conditions induced by H2O2 increased the levels of CsMb transcripts in a dose-dependent manner. Furthermore, CsMb catalyzed oxidation reactions in the presence of H2O2, and amino acid 34 of CsMb played an essential role in its reaction with H2O2. In addition, CsMb significantly reduced H2O2 and NO levels in LPS-activated macrophages, and CsMb downregulated iNOS and SOD expression in activated macrophages.ConclusionThe present study is the first to investigate the peroxidase activity of CsMb. This investigation suggested that C. sinensis may decrease the redox activation of macrophages by CsMb expression to evade host immune responses. These studies contribute to a better understanding of the role of CsMb in the molecular mechanisms involved in ROS detoxification by C. sinensis.