Sinensis Adult Worm Research Articles

Extracellular vesicles of Clonorchis sinensis promote the malignant phenotypes of cholangiocarcinoma via NF-κB/EMT axis.

Clonorchis sinensis infection is an important risk factor for cholangiocarcinoma (CCA). It has been reported that extracellular vesicles (EVs) are involved in the parasite-host interaction, and EVs of C. sinensis (CsEVs) can contribute to biliary injuries and inflammation. However, uncertainty surrounds the function of CsEVs in the progression of CCA. In this study, differential ultracentrifugation was used to separate CsEVs from the culture supernatant of C. sinensis adult worms, and they were then identified by transmission electron microscopy, nanoparticle tracking analysis and proteome assays. CCK8, EdU-488 and colony formation assays were used to explore the effect of CsEVs on the proliferation of CCA cells in vitro. Wound healing assays, transwell assays and in vivo lung metastasis model were conducted to evaluate the migration and invasion abilities. Moreover, the involvement of EMT process, as well as NF-κB and ERK signaling pathway was assessed. Results showed that CsEVs were successfully isolated and could be taken up by CCA cells, which promoted proliferation by accelerating cell cycle progression. In addition, CsEVs could facilitate cell metastasis by triggering the epithelial-mesenchymal transition (EMT). Mechanistically, activation of NF-κB signaling pathway was involved in the CsEVs-mediated EMT, which could be reversed partly by BAY 11-7082 (an inhibitor of NF-κB). In conclusion, these findings suggested that CsEVs could induce the aberrant proliferation and metastasis of CCA cells by stimulating the NF-κB/EMT axis, providing a novel theoretical explanation for liver fluke-associated CCA.

Clonorchis sinensis calcium-binding protein Cs16 causes acute hepatic injury possibly by reprogramming the metabolic pathway of bone marrow-derived monocytes.

Clonorchis sinensis infection results in various complications in the liver and biliary systems and is a neglected tropical disease in Eastern Asia. In this study, we report that C. sinensis calcium-binding protein Cs16 activates host immune cells and induces immunopathology in liver. Immunohistochemistry was used to detect the localization of Cs16 in C. sinensis adult worms. ELISA was used to detect the serum levels of anti-Cs16 IgG antibody in infected humans and mice. Bile duct injection model was used to figure out the role of Cs16 in vivo. RT-qPCR and ELISA were used to detect the cytokine production from Cs16-treated BMMs in vitro. Seahorse assay was used to detect the metabolic pathway of Cs16-treated BMMs in vitro. Cs16 localizes in the tegument and gut of C. sinensis. Humans and mice with C. sinensis infection exhibited increased levels of anti-Cs16-specific antibody. Using the bile duct injection technique, we found that Cs16 induced obvious inflammation and hepatic necrosis in vivo. Cs16 treatment caused the upregulation of inflammatory cytokines in innate immune cells. Moreover, Cs16-treated monocytes relied more on the glycolytic metabolic pathway. Our findings suggest that Cs16 is a potential pathogenic factor derived from C. sinensis adult worm. By reprogramming the metabolic pathway of innate immune cells, Cs16 triggers pro-inflammatory responses in the liver, and therefore, Cs16 is a potential target for the prevention and treatment of clonorchiasis.

Coinfection of Clonorchis sinensis and hepatitis B virus: clinical liver indices and interaction in hepatic cell models

BackgroundIn China, people infected with hepatitis B virus (HBV) are commonly found in areas with a high prevalence of Clonorchis sinensis, a trematode worm. Published studies have reported that the progression of hepatitis B is affected by coinfection C. sinensis.MethodsClinical data from a total of 72 patients with C. sinensis and HBV (as sole infection or with coinfections) and 29 healthy individuals were analysed. We also incubated the hepatic stellate cell line LX-2 with total proteins from C. sinensis adult worms (CsTPs) and HBV-positive sera. In addition, the human hepatoblastoma cell line HepG2.2.15 was treated with the antiviral drug entecavir (ETV), CsTPs and the anti-C. sinensis drug praziquantel (PZQ).ResultsOur clinical data indicated that the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB) and hyaluronic acid (HA) were significantly higher in patients with coinfection than in those infected with HBV only. In cell models, compared with the model in which LX-2 cells were incubated with HBV-positive sera (HBV group), transcripts of alpha-smooth muscle actin and types I and III collagen were significantly elevated in the models of LX-2 cells treated with CsTPs and HBV-positive sera (CsTP+HBV group), while the messenger RNA levels of tumour necrosis factor-α, interleukin (IL)-1β and IL-6 in the CsTP+HBV group were clearly lower. The HBV surface antigen and hepatitis B e-antigen levels were higher in the HepG2.2.15 cells treated with ETV and CsTPs than in those in the ETV group and in the cells administered a mixture of ETV, CsTPs and PZQ.ConclusionsThese results confirmed that C. sinensis and HBV coinfection could aggravate the progression of liver fibrosis. CsTPs might promote chronic inflammation of the liver in individuals with HBV infection, resulting in the development of hepatic fibrosis.Graphic abstract

Molecular and Structural Characterization of the Tegumental 20.6-kDa Protein in Clonorchis sinensis as a Potential Druggable Target.

The tegument, representing the membrane-bound outer surface of platyhelminth parasites, plays an important role for the regulation of the host immune response and parasite survival. A comprehensive understanding of tegumental proteins can provide drug candidates for use against helminth-associated diseases, such as clonorchiasis caused by the liver fluke Clonorchis sinensis. However, little is known regarding the physicochemical properties of C. sinensis teguments. In this study, a novel 20.6-kDa tegumental protein of the C. sinensis adult worm (CsTegu20.6) was identified and characterized by molecular and in silico methods. The complete coding sequence of 525 bp was derived from cDNA clones and encodes a protein of 175 amino acids. Homology search using BLASTX showed CsTegu20.6 identity ranging from 29% to 39% with previously-known tegumental proteins in C. sinensis. Domain analysis indicated the presence of a calcium-binding EF-hand domain containing a basic helix-loop-helix structure and a dynein light chain domain exhibiting a ferredoxin fold. We used a modified method to obtain the accurate tertiary structure of the CsTegu20.6 protein because of the unavailability of appropriate templates. The CsTegu20.6 protein sequence was split into two domains based on the disordered region, and then, the structure of each domain was modeled using I-TASSER. A final full-length structure was obtained by combining two structures and refining the whole structure. A refined CsTegu20.6 structure was used to identify a potential CsTegu20.6 inhibitor based on protein structure-compound interaction analysis. The recombinant proteins were expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid affinity chromatography. In C. sinensis, CsTegu20.6 mRNAs were abundant in adult and metacercariae, but not in the egg. Immunohistochemistry revealed that CsTegu20.6 localized to the surface of the tegument in the adult fluke. Collectively, our results contribute to a better understanding of the structural and functional characteristics of CsTegu20.6 and homologs of flukes. One compound is proposed as a putative inhibitor of CsTegu20.6 to facilitate further studies for anthelmintics.

RNAi-mediated silencing of enolase confirms its biological importance in Clonorchis sinensis

Clonorchis sinensis (C. sinensis) infection is still a common public health problem in freshwater fish consumption areas in Asian countries. More molecular evidence are required to speed up the prevention strategies to control this kind of infectious disease. In the present study, to confirm the biological importance of Csenolase followed by our previous observations of the key metabolic enzyme, we explored the RNA silence effect of the Csenolase-derived RNA interference (RNAi) in C. sinensis. The extramembranous region aa105-226 was selected as the target sequence of RNA silence. Csenolase-derived double strand RNA (dsRNA-Csenolase, 366 bp) was synthetized and delivered into C. sinensis by soaking approach. The penetration of dsRNA into adult worms and metacercariae was tracked using fluorescently labeled RNA. Western blotting and qRT-PCR experiments were performed to determine dsRNA-Csenolase-silencing effect. Our results showed that, after incubating for 120 h, dsRNA-Csenolase could effectively target and downregulate the expression of Csenolase in both adult worms (P < 0.001) and metacercariae (P < 0.01), resulting in a remarkable killing effect on C. sinensis adult worms (P < 0.01). Fluorescent Cy3-labeled dsRNA was mostly deposited in the uterus and vitellarium of adult worm and in the cyst wall of metacercaria. The present study is the first report of RNAi trials in C. sinensis, allowing further applications in identifying functional genes in C. sinensis.

Proteomic analysis of different period excretory secretory products from Clonorchis sinensis adult worms: molecular characterization, immunolocalization, and serological reactivity of two excretory secretory antigens—methionine aminopeptidase 2 and acid phosphatase

The excretory secretory products (ESP) of Clonorchis sinensis are the causative agents of clonorchiasis and biliary diseases. The parasites' ESP play important roles in host-parasite interactions. The protein compositions of ESP at different secretory times are different and have not been systemically investigated so far. In this study, we collected ESP from six different periods (0-3h, 3-6h, 6-12h, 12-24h, 24-36h, and 36-48h) from C. sinensis adults. Using a shotgun LC-MS/MS analysis, we found 187, 80, 103, 58, 248, and 383 proteins, respectively. Among these proteins, we selected methionine aminopeptidase 2 (MAP-2, presented in 24-36h and 36-48h ESP) and acid phosphatase (AP, presented in 3-6h, 12-24h, 24-36h, and 36-48h ESP) for further study. Bioinformatics analysis showed that CsMAP-2 has metallopeptidase family M24, unique lysine residue-rich and acidic residue-rich domain, SGTS motif, and auto-cleavage point; and that CsAP has possible signal sequence cleavage site, acid phosphate domain, and two histidine acid phosphatases active regions. CsMAP-2 and CsAP's cDNA have 1,425bp and1,410bp ORF, encoding 475 and 470 amino acid proteins and weighing 55.3840kDa and 55.2875kDa, respectively. MAP-2 and AP were identified as antigens present in the ESP and circulating antigens by immunoblot analysis, which were also found expressing in the eggs, metacercaria, and adult stages of C. sinensis. Immunofluorescence analysis showed that they were located in tegument and intestinal cecum of adult. MTT assay showed that they could inhibit hepatic stellate cell line (LX-2) proliferation. These findings presented the compositions of different period excretory secretary products from C. sinensis adults.

In Vitro Maintenance of Clonorchis sinensis Adult Worms

Clonorchis sinensis is a biological carcinogen inducing human cholangiocarcinoma, and clonorchiasis is one of the important endemic infectious diseases in East Asia. The present study investigated survival longevity of C. sinensis adult worms in various in vitro conditions to find the best way of keeping the worms longer. The worms were maintained in 0.85% NaCl, 1×PBS, 1×Locke's solution, RPMI-1640, DMEM, and IMDM media, and in 1×Locke's solution with different supplements. All of the worms died within 3 and 7 days in 0.85% NaCl and 1×PBS, respectively, but survived up to 57 days in 1×Locke's solution. The worms lived for 106 days in DMEM, and 114 days in both RPMI-1640 and IMDM media. The survival rate in RPMI-1640 medium was the highest (50%) compared to that in DMEM (20±10%) and in IMDM (33.3±25.2%) after 3 months. The 1×Locke's solution with 0.005% bovine bile supplement showed increased duration of maximum survival from 42 days to 70 days. Higher concentration of bile supplements than 0.005% or addition of glucose were disadvantageous for the worm survival. The worms died rapidly in solutions containing L-aspartic acid, L-glutamic acid, and adenine compared to L-arginine, L-serine, and L-tryptophan. In conclusion, the 1×Locke's solution best supports the worms alive among inorganic solutions for 57 days, and the RPMI-1640 medium maintains living C. sinensis adults better and longer up to 114 days in vitro than other media.

Gene/protein expression level, immunolocalization and binding characteristics of fatty acid binding protein from Clonorchis sinensis (CsFABP)

Clonorchis sinensis fatty acid-binding protein (CsFABP) belongs to a multigene family of lipid-binding proteins and is considered to be a promising vaccine candidate for human clonorchiasis. In this study, binding characteristics of CsFABP have been examined for the first time. The recombinant CsFABP (rCsFABP) was found to bind 11-(dansylamino) undecanoic acid (DAUDA), causing a blue shift in the fluorescence emission from 543 to 531 nm with an excitation wavelength of 345 nm and a substantial increase in fluorescence intensity. Fluorimetric titration of rCsFABP with DAUDA exhibited an apparent dissociation constant (K (d)) of 1.58 ± 0.14 μM. In the competitive experiment, the rCsFABP efficiently bound saturated C(10)-C(18) fatty acids and unsaturated fatty acids (oleic acid and linoleic acid), and the latter presented the higher affinity. Furthermore, quantitative RT-PCR and western blotting analysis revealed that CsFABP mRNA and protein were differentially expressed throughout the developmental cycle stages of the parasite, which occur in the definitive host (metacercariae, adult worms, and eggs). In addition, immunolocalization assay showed that CsFABP was localized on the vitelline gland, tegument, intestine, seminal vesicle, eggs in uterus, ovary, and testicle of C. sinensis adult worm, as well as on the vitelline gland of metacercaria. Intriguingly, the surface tissue of the bile duct where C. sinensis resided in the infected Sprague-Dawley rat was also strongly labeled, implying that CsFABP may possibly mediate direct interactions with host cells as a component of excretory/secretory products.

Proteomic analysis of excretory secretory products from Clonorchis sinensis adult worms: molecular characterization and serological reactivity of a excretory–secretory antigen-fructose-1,6-bisphosphatase

Clonorchis sinensis is a food-borne zoonotic parasite that resides in bile ducts and causes clonorchiasis, which may result in cholelithiasis, cholecystitis, hepatic fibrosis, and liver tumors. Although total excretory secretory products (ESP) of C. sinensis adults induce hepatic fibrosis in vivo in rats, the causative mechanism is not well understood. To study components of the ESP, C. sinensis culture medium was collected and analyzed using shotgun LC-MS/MS. We identified a total of 110 proteins, including glycometabolic enzymes (such as fructose-1,6-bisphosphatase (FBPase) and enolase), detoxification enzymes (such as glutamate dehydrogenase, dihydrolipoamide dehydrogenase and cathepsin B endopeptidase), and a number of RAB family proteins. To identify a potential causative agent for hepatic fibrosis, we expressed and purified a recombinant FBPase, a 1,041-bp gene product that encodes a 41.7-kDa protein with prototypical FBPase domains and that can form a tetramer with a molecular mass of 166.8kDa. In addition, we found that FBPase is an antigen present in the ESP and in circulation. Immunofluorescence showed that FBPase localizes to the intestinal cecum and vitellarium in C. sinensis adults. Our results describe the components of the excretory secretory products from C. sinensis adult worms and suggest that FBPase may be an important antigen present in the ESP of C. sinensis and may lay the foundation for additional studies on the development of clonorchiasis-associated hepatic fibrosis.

Identification of a serodiagnostic antigen, legumain, by immunoproteomic analysis of excretory‐secretory products of Clonorchis sinensis adult worms

Clonorchis sinensis, the Chinese liver fluke, is the causative agent of clonorchiasis as well as liver and biliary diseases. The excretory-secretory products (ESPs) of the parasites play important roles in host-parasite interactions. In this study, we have investigated the proteome of ESPs obtained from C. sinensis adult worms. Although the full genome database of C. sinensis is not yet available, we have successfully identified 62 protein spots using 2-DE-based mass analysis and EST database of C. sinensis. The proteins identified include detoxification enzymes, such as glutathione S-transferase and thioredoxin peroxidase, myoglobin and a number of cysteine proteases that are expressed abundantly. In order to identify potential targets for the diagnosis and therapy of clonorchiasis, we conducted immunoblot analysis of the ESPs proteome using the sera obtained from clonorchiasis patients and identified legumains and cysteine proteases as antigens present in the ESPs. Although the cysteine proteases were previously reported to elicit antigenicity, the legumains are found herein for the first time as a serological antigen of C. sinensis. To confirm these findings, we expressed recombinant legumain in Escherichia coli and verified that recombinant legumain also functions as a potent antigen against the sera of clonorchiasis patients. Our results illustrate the validity of immuno-proteomic approaches in the identification of serodiagnostic antigens in the parasites.

Collection of Clonorchis sinensis adult worms from infected humans after praziquantel treatment

A cohort was established for evaluation of cancer risk factors in Sancheong-gun, Gyeongsangnam-do, Korea. As one of the cohort studies, stools of 947 residents (403 males and 544 females, age range: 29-86 years) were screened for Clonorchis sinensis eggs using both Kato-Katz method and formalin-ether sedimentation technique. The overall egg positive rate of C. sinensis was 37.7% and individual EPG (eggs per gram of feces) counts ranged from 24 to 28,800. Eight egg positive residents voluntarily joined a process of collection of the passed worms after praziquantel treatment. A total of 158 worms were recovered from 5 of the 8 treated persons, ranged from 3 to 108 in each individual. The worms were 15-20 mm x 2-3 mm in size, and showed brown-pigmented, red, or white body colors. This is the first collection record of C. sinensis adult worms from humans through anthelmintic treatment and purgation. The adult worms of C. sinensis may be paralyzed by praziquantel and then discharged passively through bile flow in the bile duct and by peristaltic movement of the bowel.

Molecular cloning and characterization of a novel lactate dehydrogenase gene from Clonorchis sinensis

From a Clonorchis sinensis adult worm cDNA library, we isolated a cDNA clone encoding a novel lactate dehydrogenase (LDH) gene which encoded a putative protein with a predicted molecular weight of 35.6 kDa. The optimum pH and temperature for the enzyme were 7.5 and 50 degrees C in the pyruvate reduction while 11 and 80 degrees C in the lactate oxidation reaction, respectively. CsLDH showed no substrate inhibition by high lactate and NAD(+) concentration, and the optimal pyruvate and optimal NADH concentrations were 10 and 0.5 mmol/l, respectively. The relative activities of these 2-oxocarboxylic acids were pyruvic acid>2-ketobutyrate>oxalacetic acid>alpha-ketoglutaric acid>phenylpyruvate. The cofactor 3-acetylpyridine adenine dinucleotide was much more effective than NAD(+). The cofactor analogs in which the nicotinamide ring is replaced by 3-pyridinealdehyde were lower activity cofactors, while the nicotinamide ring is replaced by nicotinic acid or thionicotinamide which is not a cofactor to CsLDH. The succinic acid and malic acid are not substrates of CsLDH. Cu(2+), Fe(2+), and Zn(2+) greatly inhibited the CsLDH activity both in the direction of pyruvate reduction and in the direction of lactate oxidation. The inhibition of CsLDH by gossypol may make gossypol a potential therapy drug or a lead compound for C. sinensis. Accordingly, the CsLDH may be a novel potential drug target.

Molecular cloning and characterization of a novel adenylate kinase 3 gene from Clonorchis sinensis

Adenylate kinase (AK) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in living cells. AK catalyzes reversible high energy phosphoryl transfer reactions between ATP (or GTP) and AMP to generate ADP (or GDP). From a Clonorchis sinensis adult worm cDNA library, we isolated a cDNA clone encoding a novel AK3 isozyme. The 956 bp cDNA encodes a putative protein of 228 amino acids with a predicted molecular mass of 26.2 kDa. The recombinant CsAK3 protein produced in Escherichia coli can be refolded into a functional protein with AK3 activity. The optimum pH and temperature for the enzyme are 8.5 and 40 degrees C, respectively. The calculated activation energy is 56.04 kJ mol-1. The Km of the CsAK3 for AMP and GTP are 118 microM and 359 microM, respectively. CsAK3 is inhibited by Ap5A (>70% inhibition by 2.0 mM AP5A). Ap5A may be a potential lead compound acting on C. sinensis in which AK3 as a drug target.

Immunodiagnosis of clonorchiasis using a recombinant antigen.

A cDNA expression library of Clonorchis sinensis adult worm was constructed, and screened out immunologically. One clone, pBCs31, was selected in view of its predominant reactivity with an experimentally infected rabbit serum. Recombinant C. sinensis antigen with 28 kDa as a beta-galactosidase fusion protein produced in Escherichia coli was identified by immunoblot analysis. The cloned gene was composed of 16 copies of a 30 base pair repeat and an additional 320 bases. The deduced amino acid sequence of the tandem repeat was AQPPKSGDGG. On RNA slot blot analysis. C. sinensis adult worm RNA showed a positive reaction with the cloned gene. Enzyme-linked immunosorbent assay using a purified recombinant antigen of pBCs31 showed high specificity for diagnosis of clonorchiasis.

A carbohydrate antigen of Clonorchis sinensis recognized by a species-specific monoclonal antibody.

The enzyme-linked immunosorbent assay (ELISA)-inhibition test using a Clonorchis sinensis species-specific mouse monoclonal antibody (MAb), CsHyb 0605-23, showed increased specificity over the conventional ELISA used for serodiagnosis of clonorchiasis. To characterize the corresponding antigen further, the MAb was tested against polysaccharide, protein and glycolipid fractions obtained from a crude extract of C. sinensis adult worms, using chloroform, methanol and phenol extractions. Only the polysaccharide fraction was recognized by the MAb among those fractions. Mild oxidation of the antigen with sodium periodate showed decreased reactivity against the MAb. We concluded that the antigen and antigenic determinants recognized by the MAb are carbohydrates.

Comparison of TIA with ELISA for circulating antibody detection in clonorchiasis

A comparison was made of a new serological method, thin layer immunoassay (TIA), and an established method, enzyme-linked immunosorbent assay (ELISA), in the detection and quantification of antibodies in clonorchiasis. Saline extract of lyophilized Clonorchis sinensis adult worm was used as antigen, and TIA by the method of Elwing et al. (1976) and ELISA by Voller et al. (1974) were performed. Using sera from known clonorchiasis cases, 100 % of the sera tested were positive by TIA and 88.3 % by ELISA. TIA produced false positive results in 14 out of 36 cases, which were 10 amoebiasis cases, 16 paragonimiasis cases and 10 healthy controls. ELISA, however, produced a small number of false positives, 7 out of 55 cases. There was correlation between immunoglobulin G level in sera and ELISA value (correlation coefficient, 0.69), whereas no correlation between immunoglobulin G level and TIA result. The performance of TIA and ELISA was not correlated in the results using homologous antigen.