Clonorchis sinensis is a food-borne zoonotic parasite, and humans infected with the liver fluke C. sinensis constitute a serious public health problem in many parts of southeast Asia, including China, Korea, and Vietnam (Lun et al., 2005). Approximately 35 million people are infected worldwide. People infected with C. sinensis mainly digest raw fish contaminated with metacercaria. C. sinensis adults, which dwell in bile ducts, cause subclinical or clinical hepatic and biliary disease with biliary epithelial hyperplasia, periductal fibrosis, and cystic changes in the ducts and even facilitate the development of cholangiocarcinoma (CCA) (Lee et al., 1993; Olnes and Erlich, 2004; Fried et al., 2011). Both clinical observations and epidemiological evidence strongly implicate C. sinensis infection in the pathogenesis of CCA (Choi et al., 2006, 2010; Shi et al., 2017).

Excretory-secretory products (ESPs) are antigens that include a complex mixture of secreted proteins and some extracellular vesicles (EVs) secreted through the parasite’s tegument and products from oral openings or the gut of the parasite. ESPs are involved in parasite–host interactions and play key roles in the migration of C. sinensis adults to intrahepatic bile ducts and in the development of liver damage and even CCA (Kim Y. J. et al., 2009; Pak et al., 2009, 2019). Knowledge of the components of ESPs has value in rendering a diagnosis (Kang et al., 2019; Kim et al., 2019). Identification of the composition of the ESPs could provide attractive materials for the identification of causative agent candidates and new drug targets. However, the proteomic profiles of the ESPs in C. sinensis remain obscure.

The tegument constitutes the outermost surface of the parasite and is composed of a syncytial layer. The outer surface proteins of the tegument are enriched in enzymes and glycans and thus enable the parasite to directly contact immune cells, antibodies and various cytokines. Tegument proteins are thought to play key roles in parasite reproduction, signal transduction, nutrition intake, pathogenesis and the modulation of host immune responses and are therefore promising new drug targets and vaccine candidates (Dalton et al., 2004; Jones et al., 2004; Loukas et al., 2007). Previous studies have identified tegument and outer surface proteins (labeled with biotin) of Opisthorchis viverrini (Mulvenna et al., 2010b), Schistosoma mansoni (van Balkom et al., 2005; Braschi and Wilson, 2006; Braschi et al., 2006; Sotillo et al., 2015), Schistosoma japonicum (Mulvenna et al., 2010a), Streptococcus bovis (de la Torre-Escudero et al., 2013), and Fasciola hepatica (Wilson et al., 2011; Ravidà et al., 2016), and some important proteins have been revealed to be useful for immunologic diagnosis, drug targeting and vaccine development (Mossallam et al., 2015; Chen et al., 2016). Here, we characterize both the ESPs and the tegument proteins of C. sinensis through tryptic digestion coupled with high-resolution LC-MS/MS and describe the proteins that have potential roles in pathogenesis and carcinogenesis and that can serve as potential vaccine antigens and drug targets.

The genome and transcriptome of C. sinensis have been revealed (Young et al., 2010; Wang et al., 2011b; Huang et al., 2013), and this knowledge greatly contributes to our understanding of different processes, such as parasite development, nutrition, immune evasion, and modulation. The completed C. sinensis genome was sequenced and annotated, and approximately 16,000 protein-coding gene models have been predicted (Wang et al., 2011b); in addition, a large number of secreted and tegument proteins have been identified and are available in GenBank, which makes the search for C. sinensis proteins accessible and reliable. Several reports have indicated that the ESPs of C. sinensis might play key pathogenic roles in the development of hepatic and bile duct diseases (Kim H. G. et al., 2009, Kim et al., 2019). However, few studies have examined the identities of C. sinensis ESPs. Zheng et al. (2011, 2013) identified 110 and 267 proteins in C. sinensis ESPs by LC–MS/MS after culture for 0–48 h (Zheng et al., 2011) and 0–6 h (Zheng et al., 2013), respectively. In the current study, we identified 175 ESPs, and the differences in the number of identified proteins might be associated with the protocol used for protein preparation, the protein search software program and the parameter settings. In our research, we used liquid enzyme hydrolysis, not gel electrophoresis, to effectively reduce the loss of protein. In the protein search, we set a rigorous quality error for the secondary spectrum. Because not all details of the proteins identified by Zheng et al. (2011) and Zheng et al. (2013) are included in the papers and published databases, we are unable to perform an in-depth comparison. However, some of the proteins, such as enolase, actin, heat shock protein, and Ras-related protein, were found in both the previous investigations and the current study.

The ESPs of parasites include molecules that might be useful in the development of diagnostics, vaccines, and drug therapies. In the current study, a total of 175 proteins were identified after 6 h of the in vitro culture of C. sinensis, and these included some important proteins, such as enolase, which is a cytosolic glycolytic enzyme that localizes to the cell surface and tegument in helminths. In some schistosome species (S. bovis and S. japonicum), enolase has been identified at the host-parasite surface and serves as a PLMG activator (de la Torre-Escudero et al., 2010; Yang et al., 2010; Figueiredo et al., 2015). In C. sinensis, enolase is an important glycolytic enzyme required for parasite growth (Wang et al., 2011a), and knockdown of the enolase gene in C. sinensis increases worm mortality (Wang et al., 2014a). Cathepsins have been identified as good diagnostic antigens, in addition, it has been associated with nutrient uptake (Na et al., 2008; Ma et al., 2019), and several cysteine protease subtypes, such as cathepsin A, B, D, E, and F, have been found in C. sinensis (Wang et al., 2011b; Kang et al., 2019); After 6 h of C. sinensis culture, we identified cathepsin C in the ESP fraction, and cathepsin C was specifically distributed in the suckers (oral and ventral suckers), eggs, vitellarium, intestines, and testis of C. sinensis, and it plays roles in the digestion of host proteins, nutrition assimilation, and immune invasion. Furthermore, cathepsin C might be a potential diagnostic antigen and drug target against C. sinensis infection (Liang et al., 2014).

In the ESPs of flukes that should contain some EVs, EVs can be purified from the ESPs by differential ultracentrifugation, and the EVs of some trematodes have been identified from the secretory products (Marcilla et al., 2012; Chaiyadet et al., 2015; Davis et al., 2019). Previous studies have shown that EVs of trematodes contain some of the proteins previously identified as components of ESPs; specifically, more than half of the molecules identified in the secretomes of Echinostoma caproni and F. hepatica are found in these exosome-like vesicles (Marcilla et al., 2012). At present, the EVs of C. sinensis have not been studies, and we could not identify overlapping proteins between ESPs and EVs.

Previous studies have identified the tegument proteins of O. viverrini (Mulvenna et al., 2010b), F. hepatica (Wilson et al., 2011), S. japonicum (Mulvenna et al., 2010a), S. mansoni (van Balkom et al., 2005; Braschi and Wilson, 2006; Braschi et al., 2006; Sotillo et al., 2015), and S. bovis (de la Torre-Escudero et al., 2013). Using the techniques described, the WT of C. sinensis was removed by freeze-thawing and successively extracted with buffers of increasing solubilization strength, which enabled the enrichment of membrane-associated proteins, and proteins within and exposed at the tegument surface were identified. Of the 352 identified C. sinensis tegument proteins identified, many were proteases, and these proteases might be closely related to nutrient uptake, immune escape and migration. A cathepsin B-like cysteine proteinase was identified in C. sinensis, and a novel tegument cathepsin B was identified in S. mansoni (Caffrey et al., 2002). Cathepsin B-like cysteine proteinase can decompose the skin tissue and help the cercaria penetrate into skin (Wilson and Coulson, 2006). The cathepsin B-like cysteine proteinase in C. sinensis might facilitate larval migration. The identified enolases have been previously studied and are expressed during the adult worm, metacercaria, cercaria and egg life stages of C. sinensis. Enolases exhibit a significantly higher expression level at the adult stage and are deposited on the tegument of the adult worm and cyst wall of the metacercaria. Enolases might play key roles in the growth of parasites and might be promising oral vaccine candidates (Wang et al., 2011a, 2014b). Most of the identified proteases were oxidoreductases, hydrolases and transferases, which indicated that the tegument is key for nutrient uptake and energetic metabolism.

Whole fluke biotinylation is a technique that has been used to label and identify the outer surface of tegument proteins. The reagent used in this study (Sulfo-NHS-LC-biotin) can react with exposed primary amines and preferentially labels surface-exposed proteins. Previous studies used biotin labeling to identify the outer surface proteins of O. viverrini and S. mansoni (Mulvenna et al., 2010b; Sotillo et al., 2015). The outer surfaces of O. viverrini (Mulvenna et al., 2010b), S. mansoni (van Balkom et al., 2005; Braschi and Wilson, 2006; Braschi et al., 2006; Sotillo et al., 2015), S. japonicum (Mulvenna et al., 2010a), S. bovis (de la Torre-Escudero et al., 2013), and F. hepatica (Wilson et al., 2011; Ravidà et al., 2016) have been identified, and the number of identified outer surface proteins ranges from 22 to 45. Thirty proteins were identified from the biotin-labeled outer surface of C. sinensis, and various proteins, such as actin, tetraspanin, GAPDH and annexin, were found in C. sinensis, O. viverrini, S. mansoni, S. japonicum, S. bovis, and F. hepatica (Table 1), which indicates that these outer surface proteins of flukes might have similar functions. Two tetraspanins (TSP1 and TSP2) were identified in the outer surface of S. mansoni and S. japonicum and function in tegument renewal (Tran et al., 2010), and these have been found to be promising vaccine candidates to protect against schistosomiasis (Tran et al., 2010; Chen et al., 2016). Tetraspanin 2 was previously found in C. sinensis (Kim et al., 2011), but the function of tetraspanins in C. sinensis remains unknown. Annexin is another important protein identified in the C. sinensis tegument. In S. mansoni, annexin binds to the surface membranes of the tegument in a calcium-dependent manner and is considered a potential vaccine candidate (Tararam et al., 2010). Annexin B30, which is composed of four annexin repeats, was previously found in C. sinensis and is distributed in the tegument, intestine, and eggs of adult worms and in the tegument and vitellarium of metacercaria. This protein plays a role in adjusting the host immune response during C. sinensis infection (He et al., 2014).

Considering the different biological fluids in which each parasite resides, such as the blood for schistosomes and the bile ducts for O. viverrini and C. sinensis, the tegument proteins of these two different flukes are expected to show some differences, particularly in the composition of the proteins exposed to hosts, which might be relevant to the host immune response and to nutrient uptake and metabolism. Pyruvate carboxylase, succinate dehydrogenase and glycerol kinase were found only on the outer surface of C. sinensis and O. viverrini but not in Schistosoma. These three enzymes are associated with nutrition absorption and energy metabolism. Pyruvate carboxylase is an anaplerotic enzyme that plays important roles in various cellular metabolic pathways, including fatty acid synthesis, amino acid synthesis, gluconeogenesis and glucose-induced insulin secretion. This enzyme is involved in tumorigenesis in several cancers, including breast cancer, non-small cell lung cancer, glioblastoma, renal carcinoma, and gall bladder cancer (Lao-On et al., 2018). Glycerol kinase is a highly conserved enzyme that functions at the junction of lipid synthesis and carbohydrate metabolism. This transferase catalyzes the transfer of a phosphorus group from ATP to glycerol and is a critical enzyme at the junction of fat and carbohydrate metabolism. Previous studies have shown that Plasmodium falciparum glycerol kinase catalyzes the ATP-dependent phosphorylation of glycerol to glycerol-3-phosphate (Naidoo and Coetzer, 2013). Trypanosome glycerol kinase catalyzes not only the forward reaction (ATP-dependent glycerol phosphorylation) but also the reverse reaction. Glycerol kinase is important for trypanosome survival and might be a promising drug target (Balogun et al., 2017). Succinate dehydrogenase is a functional member of both the Krebs cycle and the aerobic respiratory chain. Previous studies have shown that C. sinensis succinate dehydrogenase has varying activities during treatments with pyquiton, bithionol, and menichlopholan, and these activities are associated with morphological alterations of the tegument of the flukes (Hamajima et al., 1979; Pang et al., 1990). The determination of whether these bile duct fluke-specific enzymes play a key role in C. sinensis or O. viverrini requires further study. Some of the identified surface proteins of C. sinensis, such as Ras-related protein, beta-2-syntrophin, fatty-acid amide hydrolase, solute carrier family 25 and sodium/glucose cotransporter 4, were not found in other flukes and seem to be specific to C. sinensis. The functions of these outer surface proteins in C. sinensis have not been reported and are worth further research.

In this study, the proteins that might contribute to carcinogenesis were analyzed. Granulin, which is reportedly associated with carcinogenesis caused by O. viverrini (Smout et al., 2009), was identified in the C. sinensis tegument. In mammals, granulin is able to modulate cell growth (Shoyab et al., 1990; Culouscou et al., 1993; Zhou et al., 1993), and granulin is also a helminth-derived growth factor that causes the proliferation of mammalian cells (Smout et al., 2009, 2011). In addition, granulin was previously identified in the ESP supernatant fraction of O. viverrini after 24 h of culture (Mulvenna et al., 2010b). In C. sinensis, granulin was previously identified among the ESPs and is localized to the tegument and testes of the adult worm (Wang et al., 2017). In the current study, the absence of granulin in the supernatant obtained after 6 h of culture indicated that the protein is found at a low abundance among the ESPs. In addition to granulin, thioredoxin peroxiredoxin, carbonyl reductase 1 (CBR1) and cystatin might contribute to the carcinogenic process. Thioredoxin is a growth factor (Nguyen et al., 2006) that is overexpressed in many aggressive forms of cancer (Yoo et al., 2006). In O. viverrini, thioredoxin inhibits oxidative stress-induced apoptosis of bile duct epithelial cells, which indicates that liver fluke oxidoreductase might induce cholangiocarcinogenesis (Matchimakul et al., 2015). In C. sinensis, thioredoxin is localized to the tegument, vitelline gland, intestine, and intrauterine eggs of adult worms and is involved in the immunoregulation of the host immune response (Zhou et al., 2013), but the function of thioredoxin related to carcinogenesis remains unknown. Oxidoreductase-peroxiredoxin and carbonyl reductase 1 (CBR1) have been identified among the ESPs of C. sinensis, and both are overexpressed in liver cancer. The inhibition of CBR1 activity can enhance the effectiveness and decrease the cardiotoxicity of the anticancer drug anthracycline daunorubicin (DNR) in hepatocellular carcinoma (Huang et al., 2010). Cystatin is a superfamily of cysteine protease inhibitors that participates in various physiological and pathological processes. Cystatin stimulates interferon gamma-dependent nitric oxide production by macrophages (Vray et al., 2002), and two cystatins (Stifin-1 and Stifin-2) have been identified in C. sinensis (Kang et al., 2011, 2014). Cystatin might contribute to carcinogenesis at sites of inflammation via DNA damage and subsequent malignant transformation. Other proteins, such as FKBP12-rapamycin complex-associated protein, the molecular chaperone HtpG, phosphoenolpyruvate carboxykinase and growth factor receptor-binding protein 2, are predicted to play roles in the cancer pathway, but whether these proteins contribute to liver disease and liver cancer in C. sinensis infection needs further research. Liver fluke-induced CCA is a multifactorial pathological process associated with long-term infection and live fluke material-induced inflammation, and the release of carcinogenic substances by parasites is an important factor. The identified proteins will help elucidate the potentially important molecules and mechanisms of liver fluke-induced CCA.

In the current study, we identified the ESPs and tegument proteome of C. sinensis, and these data could contribute to a more in-depth understanding of the complex parasite-host relationship, improve the diagnosis of clonorchiasis and benefit research on the pathogenesis and development of novel interventions, drugs and vaccines to control C. sinensis infection.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

The animal study was reviewed and approved by The Institutional Review Board (IRB) approval by Guangxi Institutional Review Board (GXIRB) (IRB00001594).

YS, WZ, and ZJ contributed to the study conception and design. YS, KY, AL, YY, YH, WZ, ZJ, FO, and HW contributed to the sample collection and the acquisition, analysis and interpretation of the data. YS and KY contributed to the writing of the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.