Tae Im Kim

Developmental Transcriptomic Features of the Carcinogenic Liver Fluke, Clonorchis sinensis

Clonorchis sinensis is the causative agent of the life-threatening disease endemic to China, Korea, and Vietnam. It is estimated that about 15 million people are infected with this fluke. C. sinensis provokes inflammation, epithelial hyperplasia, and periductal fibrosis in bile ducts, and may cause cholangiocarcinoma in chronically infected individuals. Accumulation of a large amount of biological information about the adult stage of this liver fluke in recent years has advanced our understanding of the pathological interplay between this parasite and its hosts. However, no developmental gene expression profiles of C. sinensis have been published. In this study, we generated gene expression profiles of three developmental stages of C. sinensis by analyzing expressed sequence tags (ESTs). Complementary DNA libraries were constructed from the adult, metacercaria, and egg developmental stages of C. sinensis. A total of 52,745 ESTs were generated and assembled into 12,830 C. sinensis assembled EST sequences, and then these assemblies were further categorized into groups according to biological functions and developmental stages. Most of the genes that were differentially expressed in the different stages were consistent with the biological and physical features of the particular developmental stage; high energy metabolism, motility and reproduction genes were differentially expressed in adults, minimal metabolism and final host adaptation genes were differentially expressed in metacercariae, and embryonic genes were differentially expressed in eggs. The higher expression of glucose transporters, proteases, and antioxidant enzymes in the adults accounts for active uptake of nutrients and defense against host immune attacks. The types of ion channels present in C. sinensis are consistent with its parasitic nature and phylogenetic placement in the tree of life. We anticipate that the transcriptomic information on essential regulators of development, bile chemotaxis, and physico-metabolic pathways in C. sinensis that presented in this study will guide further studies to identify novel drug targets and diagnostic antigens.

Reference genes for quantitative analysis on Clonorchis sinensis gene expression by real-time PCR

The accuracies of relative gene expressions as determined by quantitative real-time polymerase chain reaction are largely dependent on the variabilities of the reference genes used. Validation of the stabilities of reference genes under experimental conditions is an essential initial step for comparative studies on the expression levels of target genes in experimental groups. Using three total RNA samples extracted independently from Clonorchis sinensis metacercariae and adults, we determined the gene expression stabilities of eight reference gene candidates and the relative transcript levels of three target genes using the geNorm program. The reference genes found to be stably expressed in metacercariae and adults were phosphoglycerate kinase, β-actin, and calcyphosine; reference genes found to be stably expressed under γ-irradiated and non-irradiated conditions were succinate dehydrogenase, small nuclear ribonucleoprotein, and β-actin; and those stably expressed regardless of bile treatment were small nuclear ribonucleoprotein, phosphoglycerate kinase, and succinate dehydrogenase. According to our data, the expression levels of target genes are dependent on normalization factors, such as the CT values of single reference genes and the geometric mean of the CT values of three reference genes. When comparing C. sinensis gene expressions, we propose to employ the geometric mean of the CT values of more than three reference genes validated in the same experimental setting.

Expressed sequence tag analysis of adult Clonorchis sinensis, the Chinese liver fluke

Expressed sequence tag (EST) pools represent partial profiles of the gene expressions of organisms. In an effort to construct a Clonorchis sinensis EST pool, 2,387 ESTs were collected from an adult C. sinensis cDNA library and assembled into 1,573 clusters. Of these clusters, 1,225 ESTs (51%) were singletons and 348 clusters consisted of more than two ESTs. There were 848 clusters (54%) that shared significant identity with previously reported proteins, and of these, 401 clusters were categorized into 11 major functional protein classes. Three cDNA clones of fructose-1,6-bisphosphate (FBP) aldolase were selected from the C. sinensis EST pool and analyzed for phylogenic clustering. FBP clones encoded a complete polypeptide, which shared significant identity to those of vertebrate and invertebrate animals and clustered with those of trematodes. We believe that the EST pool described can be confidently used as a platform in multigene researches on C. sinensis gene expression.

Functional Genes and Proteins of Clonorchis sinensis

During the past several decades, researches on parasite genetics have progressed from biochemical and serodiagnostic studies to protein chemistry, molecular biology, and functional gene studies. Nowadays, bioinformatics, genomics, and proteomics approaches are being applied by Korean parasitology researchers. As for Clonorchis sinensis, investigations have been carried out to identify its functional genes using forward and reverse genetic approaches and to characterize the biochemical and biological properties of its gene products. The authors review the proteins of cloned genes, which include antigenic proteins, physiologic and metabolic enzymes, and the gene expression profile of Clonorchis sinensis.

Tracing of the Bile-chemotactic migration of juvenile Clonorchis sinensis in rabbits by PET-CT.

Background Adult Clonorchis sinensis live in the bile duct and cause clonorchiasis. It is known that the C. sinensis metacercariae excyst in the duodenum and migrate up to the bile duct through the common bile duct. However, no direct evidence is available on the in vivo migration of newly excysted C. sinensis juveniles (CsNEJs). Advanced imaging technologies now allow the in vivo migration and localization to be visualized. In the present study, we sought to determine how sensitively CsNEJs respond to bile and how fast they migrate to the intrahepatic bile duct using PET-CT. Methodology/Principal Findings CsNEJs were radiolabeled with 18F-fluorodeoxyglucose (18F-FDG). Rabbits with a gallbladder contraction response to cholecystokinin-8 (CCK-8) injection were pre-screened using cholescintigraphy. In these rabbits, gallbladders contracted by 50% in volume at an average of 11.5 min post-injection. The four rabbits examined were kept anesthetized and a catheter inserted into the mid duodenum. Gallbladder contraction was stimulated by injecting CCK-8 (20 ng/kg every minute) over the experiment. Anatomical images were acquired by CT initially and dynamic PET was then carried out for 90 min with a 3-min acquisition per frame. Twelve minutes after CCK-8 injection, about 3,000 18F-FDG-labeled CsNEJs were inoculated into the mid duodenum through the catheter. Photon signals were detected in the liver 7–9 min after CsNEJs inoculation, and these then increased in the whole liver with stronger intensity in the central area, presenting that the CsNEJs were arriving at the intrahepatic bile ducts. Conclusion In the duodenum, CsNEJs immediately sense bile and migrate quickly with bile-chemotaxis to reach the intrahepatic bile ducts by way of the ampulla of Vater.

Identification and Molecular Characterization of Parkin in Clonorchis sinensis

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn2+ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.

Bile components and amino acids affect survival of the newly excysted juvenile Clonorchis sinensis in maintaining media

Clonorchis sinensis thrives on bile juice. The effects of bile and bile acids on newly excysted juvenile C. sinensis (CsNEJ) were studied in terms of survival. Survival of CsNEJs maintained in 1× Locke’s solution, Dulbecco’s modified Eagle’s medium, NCTC 109, Eagle’s, RPMI 1640, and 0.1% glucose was high, but dropped rapidly in 2× Locke’s, 0.85% NaCl, and phosphate-buffered saline. Most amino acids in the media favored CsNEJ survival; however, aspartic and glutamic acids and adenine reduced survival. Survival was also significantly lower in media containing more than 0.1% bile. CsNEJs preconditioned in low bile media survived longer in higher bile media. All bile acids and conjugated bile salts were found to favor CsNEJ survival, except for lithocholic acid (LCA) which was toxic. NCTC 109 medium was found to be optimal for the in vitro maintenance of CsNEJs and 1× Locke’s solution to be suitable for analyzing the biological effects of bioactive compounds and molecules. Based on these results, we propose that bile acids enhance activity of CsNEJs, but LCA deteriorate CsNEJs.

Comparison of the diagnostic performance of microscopic examination with nested polymerase chain reaction for optimum malaria diagnosis in Upper Myanmar

BackgroundAccurate diagnosis of Plasmodium infection is crucial for prompt malaria treatment and surveillance. Microscopic examination has been widely applied as the gold standard for malaria diagnosis in most part of malaria endemic areas, but its diagnostic value has been questioned, particularly in submicroscopic malaria. In this study, the diagnostic performance of microscopic examination and nested polymerase chain reaction (PCR) was evaluated to establish optimal malaria diagnosis method in Myanmar.MethodsA total of 1125 blood samples collected from residents in the villages and towns located in Naung Cho, Pyin Oo Lwin, Tha Beik Kyin townships and Mandalay of Upper Myanmar were screened by microscopic examination and species-specific nested PCR method.ResultsAmong the 1125 blood samples, 261 samples were confirmed to be infected with malaria by microscopic examination. Evaluation of the 1125 samples by species-specific nested PCR analysis revealed that the agreement between microscopic examination and nested PCR was 87.3% (261/299). Nested PCR successfully detected 38 Plasmodium falciparum or Plasmodium vivax infections, which were missed in microscopic examination. Microscopic examinations also either misdiagnosed the infected Plasmodium species, or did not detect mixed infections with different Plasmodium species in 31 cases.ConclusionsThe nested PCR method is more reliable than conventional microscopic examination for the diagnosis of malaria infections, and this is particularly true in cases of mixed infections and submicroscopic infections. Given the observed higher sensitivity and specificity of nested PCR, the molecular method holds enormous promise in malaria diagnosis and species differentiation, and can be applied as an effective monitoring tool for malaria surveillance, control and elimination in Myanmar.

Bile-induced genes in Clonorchis sinensis metacercariae

Bile stimulates many intestinal parasites, and newly excysted juvenile Clonorchis sinensis (CsNEJ) responds chemotactically to bile and matures in the bile duct. In this study, using annealing control primer-based polymerase chain reaction (PCR), 16 differentially expressed genes (DEGs) were found to be upregulated in C. sinensis metacercariae incubated in bile. Using contigs retrieved from a C. sinensis-expressed sequence tag pool, DEG sequences were extended further by DNA-walking. Of these, five DEGs were annotated to functional genes and confirmed to have been upregulated by more than twofold by quantitative real-time PCR. The gene products of these DEGs were cytochrome c oxidase subunit 2, phosphoenolpyruvate carboxykinase 2, and mitochondrial phosphate carrier protein, which are involved in energy generation, and HLA-B-associated transcript 3 and zinc finger protein, which are regulatory proteins associated with apoptosis and/or proliferation signaling pathways. Based on these results, it is suggested that bile stimulates the expressions of genes that produce the energy required by CsNEJs to migrate to the bile duct and to modulate the regulatory signals of cell proliferation associated with adult development.

Partner proteins that interact with Clonorchis sinensis WD40-repeat protein.

WD40-repeat proteins have four to eight repeat units, which have Gly–His (GH) and Trp–Asp (WD) at both termini and fold into a beta-propeller. In particular, the WD40-repeat protein of Clonorchis sinensis (CsWD1) has seven WD-repeat units and is expressed stage-specifically in metacercariae. By yeast two-hybrid screening, putative interacting protein cDNAs were cloned from a C. sinensis metacercaria cDNA library and purified further by higher stringency screening and lacZ colony-lift assay. After assessing their nucleotide and polypeptide sequences, 21 putative partner protein cDNAs were selected and assembled into 14 clones. Using YRG2 strain yeast, 12 putative partner protein clones were confirmed to interact with CsWD1 protein. These 12 proteins were grouped into functional categories, i.e., signal proteins, transporters, proteases, and muscle proteins. These results suggest that CsWD1 protein is associated with intracellular protein translocation and cell cycle control in C. sinensis metacercaria.