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

Ren, Mengyu, He, Lei, Huang, Yan, Mao, Qiang, Li, Shan, Qu, Honglin, Bian, Meng, Liang, Pei, Chen, Xueqing, Ling, Jinsi, Chen, Tingjing, Liang, Chi, Wang, Xiaoyun, Li, Xuerong, Yu, Xinbing
Parasites & vectors 2014 v.7 no.1 pp. 250
Clonorchis sinensis, Escherichia coli, adults, amino acids, bioinformatics, coculture, excretory-secretory products, foodborne illness, hepatoma, hydrogen peroxide, immune response, inducible nitric oxide synthase, macrophages, manganese, messenger RNA, mutants, myoglobin, nitric oxide, oxidation, oxidative stress, oxygen, peroxidase, reverse transcriptase polymerase chain reaction, superoxide dismutase
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 Cs Mb and its putative roles in the interactions of C. sinensis with its host, we explored the molecular characterization of Cs Mb in this paper. METHODS: We expressed Cs Mb 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 Cs Mb was confirmed by spectrophotometry. We co-cultured RAW264.7 cells with recombinant Cs Mb (rCs Mb), 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 Cs Mb increased with the increase of oxygen content. Oxidative stress conditions induced by H₂O₂ increased the levels of Cs Mb transcripts in a dose-dependent manner. Furthermore, Cs Mb catalyzed oxidation reactions in the presence of H₂O₂, and amino acid 34 of Cs Mb played an essential role in its reaction with H₂O₂. In addition, Cs Mb significantly reduced H₂O₂ and NO levels in LPS-activated macrophages, and Cs Mb downregulated iNOS and SOD expression in activated macrophages. CONCLUSION: The present study is the first to investigate the peroxidase activity of Cs Mb. This investigation suggested that C. sinensis may decrease the redox activation of macrophages by Cs Mb expression to evade host immune responses. These studies contribute to a better understanding of the role of Cs Mb in the molecular mechanisms involved in ROS detoxification by C. sinensis.