Identification and molecular characterization of a novel signaling molecule 14-3-3 epsilon in Clonorchis sinensis excretory/secretory products.

Increasing evidence shows that 14-3-3 proteins are involved in many biology events in addition to signal transduction. Extensive investigations on structural and biochemical features of these signaling molecules have implied their importance in the biological process. In the present study, we have identified and characterized the 14-3-3 epsilon (Cs14-3-3) in Clonorchis sinensis that causes human clonorchiasis. Recombinant protein was expressed in Escherichia coli (E. coli) and identified by MALDI-TOF/TOF. Immunoblot results revealed that Cs14-3-3 was a component of excretory/secretory products. Ligand blot assay indicated that 14-3-3 epsilon could bind C. sinensis MAPKAPK 2 in a nonphosphorylation-dependent manner. This protein could be detected at four stages of the life cycle by RT-PCR experiments and immunolocalization showed that Cs14-3-3 was extensively distributed in C. sinensis, especially at the outer surface and the sucker of adult worm and cyst wall of metacercaria. Taken together, 14-3-3 epsilon might play some roles in the development of the parasites. In addition, Cs14-3-3 epsilon should be addressed for the diagnostic value in C. sinensis infection in consideration of high sensitivity and specificity. As an immune stimulus, C. sinensis 14-3-3 epsilon was found to provoke a Th1/Th2 balanced immune response by inducing high levels of both IgG1 and IgG2a. Recombinant Cs14-3-3 conferred effective protection both in worm reduction rate and egg reduction rate, suggesting that the signaling molecule Cs14-3-3 was a promising vaccine candidate against C. sinensis infection.

The draft genome of the carcinogenic human liver fluke Clonorchis sinensis.

BACKGROUND: Clonorchis sinensis is a carcinogenic human liver fluke that is widespread in Asian countries. Increasing infection rates of this neglected tropical disease are leading to negative economic and public health consequences in affected regions. Experimental and epidemiological studies have shown a strong association between the incidence of cholangiocarcinoma and the infection rate of C. sinensis. To aid research into this organism, we have sequenced its genome. RESULTS: We combined de novo sequencing with computational techniques to provide new information about the biology of this liver fluke. The assembled genome has a total size of 516 Mb with a scaffold N50 length of 42 kb. Approximately 16,000 reliable protein-coding gene models were predicted. Genes for the complete pathways for glycolysis, the Krebs cycle and fatty acid metabolism were found, but key genes involved in fatty acid biosynthesis are missing from the genome, reflecting the parasitic lifestyle of a liver fluke that receives lipids from the bile of its host. We also identified pathogenic molecules that may contribute to liver fluke-induced hepatobiliary diseases. Large proteins such as multifunctional secreted proteases and tegumental proteins were identified as potential targets for the development of drugs and vaccines. CONCLUSIONS: This study provides valuable genomic information about the human liver fluke C. sinensis and adds to our knowledge on the biology of the parasite. The draft genome will serve as a platform to develop new strategies for parasite control.

Molecular characterization of cathepsin B from Clonorchis sinensis excretory/secretory products and assessment of its potential for serodiagnosis of clonorchiasis.

BACKGROUND: Cathepsin cysteine proteases play multiple roles in the life cycle of parasites such as food uptake, immune invasion and pathogenesis, making them valuable targets for diagnostic assays, vaccines and drugs. The purpose of this study was to identify a cathepsin B of Clonorchis sinensis (CsCB) and to investigate its diagnostic value for human helminthiases. RESULTS: The predicted amino acid sequence of the cathepsin B of C. sinensis shared 63%, 52%, 50% identity with that of Schistosoma japonicum, Homo sapiens and Fasciola hepatica, respectively. Sequence encoding proenzyme of CsCB was overexpressed in Escherichia coli. Reverse transcription PCR experiments revealed that CsCB transcribed in both adult worm and metacercaria of C. sinensis. CsCB was identified as a C. sinensis excretory/secretory product by immunoblot assay, which was consistent with immunohistochemical localization showing that CsCB was especially expressed in the intestine of C. sinensis adults. Both ELISA and western blotting analysis showed recombinant CsCB could react with human sera from clonorchiasis and other helminthiases. CONCLUSIONS: Our findings revealed that secreted CsCB may play an important role in the biology of C. sinensis and could be a diagnostic candidate for helminthiases.

Molecular cloning, expression, and immunolocalization of the NAD(+)-dependent glycerol 3-phosphate dehydrogenase (GPD) from Clonorchis sinensis.

Glycerol 3-phosphate dehydrogenase (GPD) plays an important role in the energy metabolism and nutrition metabolism. In order to know about the biological functions of GPD of Clonorchis sinensis (C. sinensis), we identified a complete gene coding GPD from C. sinensis metacercaria cDNA library. This novel cDNA sequence contains 1,056 bp with a putative open reading frame of 351 amino acids and shares 74% identity with GPD from Schistosoma mansoni. Recombinant CsGPD was expressed and purified from Escherichia coli BL21 (DE3). Western blot analysis displayed that recombinant CsGPD can be recognized by anti-CsGPD serum and C. sinensis-infected serum. RT-PCR and immunolocalization analysis confirmed that GPD expressed both at the stage of adult worm and metacercaria of C. sinensis and immunolocated at the tegument of adult worm, tegument and tegumentary cells of metacercaria. Our current study has paved the way for the further researches about the biological functions involved in the growth of C. sinensis.

Clonorchis sinensis enolase: identification and biochemical characterization of a glycolytic enzyme from excretory/secretory products.

Enolase plays a key role in energy metabolism and development of most organisms. We isolated a gene encoding enolase from Clonorchis sinensis (C. sinensis) adult cDNA library and expressed the recombinant protein in Escherichia coli. C. sinensis enolase (Csenolase) was identified as both an excretory/secretory product and a tegumental component of C. sinensis by western blot analysis. The transcriptional level of Csenolase was examined at adult worm, metacercaria, cercaria and egg of C. sinensis, and results showed that Csenolase is transcribed at the four life stages of C. sinensis while showing a significant higher expression level at the stage of adult worm. Immunohistochemical localization indicated that Csenolase was specifically deposited on the tegument of adult worm and cyst wall of metacercaria. Ligand blot assay revealed a specific characteristic of dose-dependent plasminogen-binding activity of Csenolase and kinetic parameters were explored using 2-phospho-D-glycerate (2-PGA) as the primary substrate by monitoring the conversion of nicotinamide-adenine dinucleotide (NADH) into nicotinamide adenine dinucleotide (NAD). In addition, Csenolase exhibited active enzyme activity in catalytic reactions while the anti-Csenolase serum inhibited the enzyme activity. In vitro incubation experiments revealed that Csenolase might play key roles in the growth of the parasites. In conclusion, Csenolase is an important glycolytic enzyme required for the development of C. sinensis, and may be a potential vaccine candidate and drug target against C. sinensis infection.

Molecular cloning and characterization of a novel ras-related protein (rap2) from Clonorchis sinensis.

Ras are key components of diverse signal transduction pathways and play important roles in growth and development. To know about growth regulation in Clonorchis sinensis, we have identified a full-length sequence encoding a ras-related protein (rap2) from our adult cDNA library. The open reading frame contains 561 bp encoding 186 amino acids. The hypothetical amino acid sequence shared high identities with rap2 proteins from Schistosoma japonicum and Homo sapiens. Conserved domains of small guanosine triphosphate-binding proteins and characteristic amino acid residues of rap2 proteins were observed in this sequence. Reverse transcription polymerase chain reaction experiments revealed that rap2 transcribed in adult worm, metacercaria, and eggs of C. sinensis. Recombinant rap2 protein was expressed and purified from Escherichia coli. rap2 could be probed by C. sinensis-infected rat serum in western blotting experiment. By immunohistochemistry, rap2 was localized on the tegument of adult worm and metacercaria of C. sinensis. This fundamental study might contribute to further researches in signaling systems that are related to growth control and development of C. sinensis and other parasites.

Experimental model in rats for study on transmission dynamics and evaluation of Clonorchis sinensis infection immunologically, morphologically, and pathologically.

This study aims to gain a better insight into the transmission patterns and immunologic profile of Clonorchis sinensis infection and make a headway on the pathogenesis regarding cholangiocarcinoma and hepatic lesions. Experimental models orally infected by C . sinensis metacercariae were constructed in rats. Immunological assays were performed to measure serum level of IgA, IgE, IgG1, IgG2a, IFN-gamma, and IL-4. Infection parameters were assessed by worm recovery rate, eggs per gram faece and worm size. Pathological sections with livers were managed with immunofluorescence, hematoxylin, eosin, and Masson's trichrome staining to evaluate the hepatic pathological changes. Interestingly, rats infected with only one C . sinensis metacercariae even gained a high worm recovery rate of 83.3% compared with rats infected with more metacercariae. Serological changes according to different infection doses indicated that immune response presented a tendency to Th2 type by expressing transient high level of IgG1, IL-4, and IgE. Hepatic tissues appeared inflammatory and fibrotic, revealed by different stainings. Intrahepatic bile ducts displayed cholangiectasis and proliferation with excreted/secreted antigen histologically located. C . sinensis, as a fish-borne zoonosis, presented novel transmission patterns which explained high infection rate in endemic areas; infection rate of C . sinensis was frequency-dependent and dose-related. Humoral immunity played a prevalent role in resisting to C . sinensis based on the rat models. C . sinensis infection played an undoubted role in biliary and hepatic diseases.