[show abstract][hide abstract] ABSTRACT: One cDNA clone (Cs18h09) encoding Clonorchis sinensis calmodulin (CsCaM) was isolated from our adult cDNA plasmid library. The open reading frame of CsCaM contains 450 bp which encodes 149 amino acids. CsCaM protein comprises four calcium-binding EF-hand motifs. The amino acid sequence of CsCaM shares very high homology with other species. Quantitative RT-polymerase chain reaction (PCR) revealed that CsCaM mRNA was constitutively transcribed in development cycle stages of the parasite, including adult worm, metacercaria, excysted metacercaria, and egg. In addition, recombinant CsCaM (rCsCaM) was expressed as a soluble protein and anti-rCsCaM rat serum could detect CsCaM in the C. sinensis somatic extracts but not in the C. sinensis excretory-secretory products (ESPs). Moreover, immunolocalization assay showed that CsCaM was located in tegument, intestine, pharynx, and eggs. Furthermore, rCsCaM was found to bind calcium ion (Ca(2+)) and magnesium (Mg(2+)) in electrophoretic mobility shift assay. Ca(2+) binding increased the ability of rCsCaM to bind the hydrophobic fluorescent probe 8-anilinonaphthalene-1-sulphonate, causing a blue shift in the fluorescence emission from 540 to 515 nm with an excitation wavelength of 380 nm and substantial increase in fluorescence intensity but not Mg(2+). Collectively, here we showed the basic characterization of CsCaM and inferred that CsCaM could be a Ca(2+) sensor protein, and CsCaM may possibly participate in growth and development of adult worm and egg of C. sinensis through binding Ca(2+).
Parasitology Research 02/2013; · 2.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Thioredoxin transmembrane related protein (TMX), a member of thioredoxin superfamily, is localized to the endoplasmic reticulum and possesses a thioredoxin-like domain that plays an important role as an oxidoreductase. The functions of TMX in Clonorchis sinensis remain to be elucidated. In this study, we cloned and characterized a novel TMX of C. sinensis (CsTMX). The CsTMX cDNA sequence contained a 414-nucleotide open-reading frame encoding a protein of 137 amino acids. A thioredoxin domain was found in the position of aa(21-117) and contained the putative active-site motif Cys-Pro-Ala-Cys. BLASTx analysis showed that CsTMX shared 39-57 % amino acid identities with TMX of other organisms. Quantitative RT-PCR analysis demonstrated that CsTMX was differentially transcribed, with the highest level of expression in the adult worm stage and the lowest expression in egg stage. In addition, immunofluorescence assay showed CsTMX was localized in the tegument, vitelline gland, intestine, and intrauterine eggs of adult worm. Besides, immunoblot assay revealed that the recombinant CsTMX (rCsTMX) could be recognized by the sera from rats infected with C. sinensis and the sera from rats immunized by excretory-secretory products. Furthermore, analysis of the antibody isotype profile revealed that rats subcutaneously immunized with rCsTMX developed rCsTMX-specific antibody, which is dominance of IgG2a in sera. Meanwhile, production of IFN-γ was elevated strongly in the supernatants of spleen cell. The results collectively indicated that CsTMX might play an important role in the host-parasite interaction, as well as CsTMX probably involved in immunoregulation of host by inducing Th1-type dominated immune response in rats.
Parasitology Research 02/2013; · 2.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Clonorchis sinensis, an ancient parasite that infects a number of piscivorous mammals, attracts significant public health interest due to zoonotic exposure risks in Asia. The available studies are insufficient to reflect the prevalence, geographic distribution, and intraspecific genetic diversity of C. sinensis in endemic areas. Here, a multilocus analysis based on eight genes (ITS1, act, tub, ef-1a, cox1, cox3, nad4 and nad5 [4.986 kb]) was employed to explore the intra-species genetic construction of C. sinensis in China. Two hundred and fifty-six C. sinensis isolates were obtained from environmental reservoirs from 17 provinces of China. A total of 254 recognized Multilocus Types (MSTs) showed high diversity among these isolates using multilocus analysis. The comparison analysis of nuclear and mitochondrial phylogeny supports separate clusters in a nuclear dendrogram. Genetic differentiation analysis of three clusters (A, B, and C) showed low divergence within populations. Most isolates from clusters B and C are geographically limited to central China, while cluster A is extraordinarily genetically diverse. Further genetic analyses between different geographic distributions, water bodies and hosts support the low population divergence. The latter haplotype analyses were consistent with the phylogenetic and genetic differentiation results. A recombination network based on concatenated sequences showed a concentrated linkage recombination population in cox1, cox3, nad4 and nad5, with spatial structuring in ITS1. Coupled with the history record and archaeological evidence of C. sinensis infection in mummified desiccated feces, these data point to an ancient origin of C. sinensis in China. In conclusion, we present a likely phylogenetic structure of the C. sinensis population in mainland China, highlighting its possible tendency for biogeographic expansion. Meanwhile, ITS1 was found to be an effective marker for tracking C. sinensis infection worldwide. Thus, the present study improves our understanding of the global epidemiology and evolution of C. sinensis.
PLoS ONE 01/2013; 8(6):e67006. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Clonorchis sinensis (C. sinensis), an important food-borne parasite that inhabits the intrahepatic bile duct and causes clonorchiasis, is of interest to both the public health field and the scientific research community. To learn more about the migration, parasitism and pathogenesis of C. sinensis at the molecular level, the present study developed an upgraded genomic assembly and annotation by sequencing paired-end and mate-paired libraries. We also performed transcriptome sequence analyses on multiple C. sinensis tissues (sucker, muscle, ovary and testis). Genes encoding molecules involved in responses to stimuli and muscle-related development were abundantly expressed in the oral sucker. Compared with other species, genes encoding molecules that facilitate the recognition and transport of cholesterol were observed in high copy numbers in the genome and were highly expressed in the oral sucker. Genes encoding transporters for fatty acids, glucose, amino acids and oxygen were also highly expressed, along with other molecules involved in metabolizing these substrates. All genes involved in energy metabolism pathways, including the β-oxidation of fatty acids, the citrate cycle, oxidative phosphorylation, and fumarate reduction, were expressed in the adults. Finally, we also provide valuable insights into the mechanism underlying the process of pathogenesis by characterizing the secretome of C. sinensis. The characterization and elaborate analysis of the upgraded genome and the tissue transcriptomes not only form a detailed and fundamental C. sinensis resource but also provide novel insights into the physiology and pathogenesis of C. sinensis. We anticipate that this work will aid the development of innovative strategies for the prevention and control of clonorchiasis.
PLoS ONE 01/2013; 8(1):e54732. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human clonorchiasis has been increasingly prevalent in recent years and results in a threat to the public health in epidemic regions, motivating current strategies of vaccines to combat Clonorchis sinensis (C. sinensis). In this study, we identified C. sinensis paramyosin (CsPmy) from the cyst wall proteins of metacercariae by proteomic approaches and characterized the expressed recombinant pET-26b-CsPmy protein (101 kDa). Bioinformatics analysis indicated that full-length sequences of paramyosin are conserved in helminthes and numerous B-cell/T-cell epitopes were predicted in amino acid sequence of CsPmy. Western blot analysis showed that CsPmy was expressed at four life stages of C. sinensis, both cyst wall proteins and soluble tegumental components could be probed by anti-CsPmy serum. Moreover, immunolocalization results revealed that CsPmy was specifically localized at cyst wall and excretory bladder of metacercaria, as well as the tegument, oral sucker and vitellarium of adult worm. Both immunoblot and immunolocalization results demonstrated that CsPmy was highly expressed at the stage of adult worm, metacercariae and cercaria, which could be supported by real-time PCR analysis. Both recombinant protein and nucleic acid of CsPmy showed strong immunogenicity in rats and induced combined Th1/Th2 immune responses, which were reflected by continuous high level of antibody titers and increased level of IgG1/IgG2a subtypes in serum. In vaccine trials, comparing with control groups, both CsPmy protein and DNA vaccine exhibited protective effect with significant worm reduction rate of 54.3% (p<0.05) and 36.1% (p<0.05), respectively. In consistence with immune responses in sera, elevated level of cytokines IFN-γ and IL-4 in splenocytes suggested that CsPmy could induce combined cellular immunity and humoral immunity in host. Taken together, CsPmy could be a promising vaccine candidate in the prevention of C. sinensis regarding its high immunogenicity and surface localization.
PLoS ONE 01/2012; 7(3):e33703. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cysteine proteases play essential roles in parasite physiology as well as in host-parasite interactions through their modulation of various biological and pathobiological events. In the present study, a full-length sequence encoding cysteine protease of Clonorchis sinensis (CsCP) was isolated from our adult cDNA library. The open reading frame contains 984 bp encoding 327 amino acids. The present amino acid sequence shared 68% identity with two known CsCP genes and 29-49% identity with that of other species. Bioinformatics analysis showed that conserved domains and characteristic amino acid residues of cysteine proteases were observed in this sequence. Real-time PCR experiments revealed that CsCP was consecutively transcribed in various developmental stages of the parasite, including adult worm, excysted juvenile, metacercaria and egg. Recombinant CsCP (rCsCP) could be probed by rat anti-CsCP serum, rabbit anti-excretory-secretory products (ESP) serum and serum from human infected with Clonorchis sinensis in Western blot. The result of immunolocalization showed that CsCP was mainly located in the oral sucker, excretory bladder and tegument of cercariae and metacercariae, as well as the intestine of adult worm. The rCsCP-based IgG and its isotypes were all detected in sera from human infected with C. sinensis by enzyme-linked immunosorbent assay, and the level of IgG1 is the highest. The receiver-operating characteristic (ROC) analysis was used to determine the most appropriate cut-off value that yielded the high sensitivity (86.96%) and specificity (70.42%). These results revealed that CsCP may play an important role in the biology of C. sinensis and could be a diagnostic candidate for clonorchiasis.
Parasitology Research 12/2011; 110(6):2211-9. · 2.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
[show abstract][hide abstract] ABSTRACT: 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.
Parasitology Research 09/2011; 110(4):1411-20. · 2.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
[show abstract][hide abstract] ABSTRACT: 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 and Biochemical Parasitology 03/2011; 177(2):135-42. · 2.73 Impact Factor