Molecular characterization of Clonorchis sinensis secretory myoglobin: delineating its role in anti-oxidative survival

Abstract

Background: Clonorchiasis 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.

Methods: We 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.

Results: In 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.

Conclusion: The 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.

Figure 1

Sequences and structure comparisons between Clonorchis sinensis myoglobin ( Cs Mb) and its orthologues. (A) The amino acid sequence of CsMb was aligned with those of related proteins, which were retrieved from the GenBank database by BLAST searches (Sequence information shown in Additional file 1: Table S1). The red boxes indicate active tyrosine. (B) The tertiary structure of CsMb was simulated by the Discovery Studio 3.5 Client program and compared with P. epiclitum hemoglobin (Protein Data Bank id: 1KFR; 40% identity). Regions around the active site are shown in yellow indicating the positions of critical amino acids (red boxes in panel A). (C) Phylogenetic analysis. The majority-role consensus tree was derived from a neighbor-joining tree of the amino acid alignment, which was constructed by the PHYLIP program. The homologues from euryarchaeota were included in the root of the tree. Arabic numerals at branching nodes indicate their percentages of appearance in 1000 bootstrap replicates.

Figure 2

Immunolocalization of CsMb in adult worm and metacercaria of C. sinensis. Rat anti-rCsMb serum was used as the primary antibody and red fluorescent Cy3-labeled goat anti-rat IgG as the secondary antibody. Slides were observed under white light (panel A, C, E, G, and I) or under a fluorescence microscope (panel B, D, F, H, and J). No specific fluorescence was observed in panel B or H, which was probed with serum from rats immunized with PBS as a negative control. Intensive reddish-orange fluorescent signals were observed in the subtegumental and mesenchymal tissues of the adult worm (panel D and F; ×50) as well as the vitellarium of the metacercaria (panel J; ×200). Scattered fluorescent signals were detected in the tegument of the metacercaria. T, tegument. W, cyst wall.

Figure 3

Transcriptional level of Mb in C. sinensis incubated in different oxygen contents. (A) The fold increase was calculated by comparing intensities between experimental and control groups. The CsMb transcripts were much higher in the 20% oxygen group than in the 1% oxygen group. The transcriptional level of CsMb in the 5% oxygen group was 3-fold higher than that in the 1% oxygen group. (B) Changes of Mb transcript levels in C. sinensis caused by oxidative chemicals. The worms were stimulated with H₂O₂ (0–1.8 mM) for 1 h at 37 °C. CsMb transcript amounts were significantly increased at H₂O₂ concentrations greater than 0.6 mM. (*p < 0.01).

Figure 4

Peroxidase activity of r Cs Mb and mutants evaluated by spectrophotometry. Reactions were performed with H₂O₂ (20 μM) and oxygenated rCsMb solutions at 20 °C and pH 7.0. The A₀/A ratio is the initial absorbance and final absorbance at 407 nm. All mutants containing Y34A did not show H₂O₂ reactivity, and only Y68A exhibited lower reactivity than that of wild-type CsMb.

Figure 5

Production of H₂O₂ and NO in activated RAW264.7 cells in various conditions. In the respective experimental conditions, the H₂O₂ level was measured after 2 h (panel A), and the NO level was measured after 24 h (panel B). LPS (100 ng/ml) was used as a positive control. The levels of H₂O₂ and NO were significantly decreased in rCsMb-treated cells compared to the levels of cells treated with LPS + PBS. * indicates p < 0.01.

Figure 6

mRNA levels of various genes involved in the oxidative burst in RAW264.7 cells. The relative mRNA level is represented by the ratio of mRNAs to β-actin. The mRNA levels were significantly downregulated in cells treated with rCsMb compared the levels of cells treated with LPS + PBS. (*p < 0.01).