Molecular characterization and serodiagnosis analysis of a novel lysophospholipase from <Emphasis Type="Italic">Clonorchis sinensis</Emphasis>

A cDNA clone encoding a novel lysophospholipase with a predicted molecular weight of 25.2 kDa was isolated from a Clonorchis sinensis adult cDNA library. The enzyme activity of the recombinant protein expressed in Escherichia coli was determined using phosphatidylcholine and lysophosphatidylcholine as substrates. Western blotting analysis indicated that it belonged to excretory/secretory proteins of the adults. The sensitivity and specificity of the recombinant antigen for serodiagnosis were evaluated with immunoglobulin enzyme-linked immunosorbent assay using serum samples from 20 patients with clonorchiasis and 20 patients with schistosomiasis. The sensitivity (75%) and specificity (80%) of the recombinant protein were comparable to those of crude extracts, at 65 and 82.5%, respectively. The sensitivity of the recombinant protein was 77% using 100 serum samples of clonorchiasis patients with various parasite burden. The results suggested that the recombinant lysophospholipase protein was not a satisfactory candidate for diagnosis of clonorchiasis, although it might be an excretory/secretory protein.     

Molecular characterization of <Emphasis Type="Italic">Clonorchis sinensis</Emphasis> tetraspanin 2 extracellular loop 2

In the course of examining EST of the liver fluke Clonorchis sinensis, a cDNA encoding the protein similar to tetraspanin 2 (TSP-2) of blood fluke schistosome was identified as CsTSP2. TSPs are a family of eukaryotic cell membrane-spanning proteins thought to anchor multiple proteins to one area of the cell membrane. Multiple sequence alignment of CsTSP2 revealed over 40% of identities with those of schistosomes. The CCC, PXSC, and CG motives characteristic to extracellular loop 2 (EC2) region of TSP-2 were well conserved. PCR-amplified EC2 of CsTSP2 (CsTSP2EC2) was subcloned into pEXP5 NT/TOPO bacterial expression plasmid vector. Recombinant CsTSP2EC2 protein (rCsTSP2EC2) fused with 6X His tag was overexpressed bacterially and purified by using Ni–NTA affinity chromatography under denaturing condition. The purified rCsTSP2EC2 was recognized specifically by the sera from human infected with C. sinensis. Considering biological role and specific immunity of TSPs, rCsTSP2EC2 might be a probable vaccine candidate against clonorchiasis. Protective effects of immunizing rCsTSP2EC2 should be further studied.     

Ultrastructural localization of phosphoglycerate kinase in adult<Emphasis Type="Italic"> Clonorchis sinensis</Emphasis>

Phosphoglycerate kinase (PGK) is an enzyme that produces one ATP molecule in the glycolytic pathway. Clonorchis sinensis is largely dependent on glycolysis for energy production. We performed immunoelectron microscopy on adult C. sinensis by using mouse immune serum raised against recombinant C. sinensis PGK. A high density of gold particles was found in the microvilli of the intestinal epithelium and in lamellae of the sperm duct. PGK was common in the somatic cells of intra-uterine eggs and in excreted products. It was localized with moderate intensity in muscular fibers of the subtegumental muscle layer, and in the myoepithelia of the intestine and excretory bladder. We suggest that PGK plays an essential role in C. sinensis energy production for movement via muscle contraction.     

<Emphasis Type="Italic">Clonorchis sinensis</Emphasis>: molecular cloning and functional expression of a novel cytosolic glutathione transferase

Glutathione transferases (GSTs) represent a large family of enzymes. In the high throughput sequencing of the cDNA library constructed from the adult stage of Clonorchis sinensis (Cs), we isolate another cDNA clone encoding a novel cytosolic GST enzyme. To discriminate with our former reported CsGST, we designated this GST as CsGST1. This new cDNA contains 744 bp with a putative open reading frame of 213 amino acids. The deduced amino acid sequence exhibits 88% identity to Opisthorchis viverrini 28GST (Ov28GST), 60 and 52% identity to C. sinensis cytosolic 28-kDa GST (Cs28GST) and CsGST, respectively. The CsGST1 was expressed in Escherichia coli BL21(DE3) as a His-tag fusion protein and was purified by Ni-NTA agarose. The recombinant CsGST1 showed moderate GST activity of 0.79 U mg⁻¹. The average K _m of the CsGST1 for 1-chloro-2, 4-dinitrobenzene is 150 μM. Cibacron blue F3GA and albendazole inhibited the CsGST1 activity with average IC₅₀ values of 9.1 and 265.4 μM, respectively. The nucleotide sequence reported in this paper was submitted to the GenBank Database with accession number DQ342327.     

Immunological cross-reactivity analysis on recombinant histamine-releasing factors from <Emphasis Type="Italic">Schistosoma japonicum</Emphasis>, <Emphasis Type="Italic">Clonorchis sinensis</Emphasis>, and Wistar rat

Studies have demonstrated a declined incidence of allergic disorders in the population with helminthic infection. Though several hypotheses have been proposed to explain how helminthic infection protected people against allergies, the underlying mechanisms remain poorly understood. A human histamine-releasing factor (HRF) has been proved to be closely related to the development of allergic disorders and the homologues are ubiquitously expressed in all eukaryotic organisms including parasites. To study the role of this HRF in the relationship between parasitic infection and allergic diseases with experimental model of rats, the cDNA of the homologues of the human HRF from Wistar rat, Schistosoma japonicum, and Clonorchis sinensis containing a coding region of 519, 510, and 510 bp, respectively, were cloned. In addition, the cross-reactivity between recombinant rat HRF (rRHRF) and recombinant S. japonicum HRF (rSjHRF) as well as that between rRHRF and recombinant C. sinensis HRF (rCsHRF) was identified with ELISA and Western blotting. Based on their detected cross-reactivities, a hypothesis was put forward that the anti-parasitic HRFs antibodies could inhibit the effects of host HRF and those of parasitic HRFs and thus decreased the host sensitivities to allergens.     

Efficacy of artesunate and artemether against <Emphasis Type="Italic">Clonorchis sinensis</Emphasis> in rabbits

Artesunate and artemether display promising clonorchicidal properties in rats. In this study, we assessed the efficacy of artesunate and artemether against Clonorchis sinensis in rabbits. Rabbits were each fed with 300 C. sinensis metacercariae. At day 28 postinfection, the rabbits were administered oral artesunate at doses of 7.5–120 mg/kg and oral artemether of 15–120 mg/kg. Two groups of rabbits were treated with single oral praziquantel at 75 and 150 mg/kg. Untreated rabbits served as controls. Fourteen days after treatment, all rabbits were sacrificed, and C. sinensis adults were collected from the bile ducts and counted. At the highest doses tested (120 mg/kg) artesunate and artemether achieved statistical significant worm burden reductions of 88.8% and 67.2%, respectively. These rates were lower than worm burden reductions observed with praziquantel (88% and 100%, respectively). It is suggested that artesunate and artemether have moderate anthelminthic efficacy against C. sinensis in rabbits.     

Cloning and expression of mitochondrial malate dehydrogenase of <Emphasis Type="Italic">Clonorchis sinensis</Emphasis>

The NAD-dependent mitochondrial malate dehydrogenase (mMDH, EC1.1.1.37) plays pivotal roles in tricarboxylic acid and is crucial for the survival and pathogenecity of parasites. A cDNA, which was identified by high throughput sequencing from the cDNA library constructed from adult Clonorchis sinensis, encoded a putative peptide of 341 amino acids with more than 50% identity with mMDHs from other organisms. The mMDH was expressed in Escherichia coli as the recombinant protein with a GST tag and purified by glutathione–Sepharose 4B column. The recombinant mMDH showed MDH activity of 63.6 U/mg, without lactate dehydrogenase activity and NADPH selectivity. The kinetic constants of recombinant mMDH were determined. Nucleotide sequence data reported in this paper are available in the GenBank under the accession number AY605670.     

Gene expression of <Emphasis Type="Italic">Clonorchis sinensis</Emphasis> metacercaria induced by gamma irradiation

Gamma-rays are a form of ionizing radiation and produce serious cellular damage to nuclei and organelles. Gamma irradiation induces the expressions of genes involved in DNA repair. Clonorchis sinensis resides in and provokes pathophysiologic changes in the bile ducts of mammals. The C. sinensis metacercariae are unsusceptible or resistant to gamma irradiation with LD₅₀ of 16.5 Gy. Using the annealing control primer-based polymerase chain reaction (PCR) method, 19 genes were found to be up-regulated in C. sinensis metacercariae exposed to gamma rays. Contigs of up-regulated genes (URGs) were retrieved in a C. sinensis expressed sequence tag pool and extended by DNA-walking. Of the 13 URGs annotated putatively as functional genes, five URGs were associated with energy metabolism, six with protein processing, and the other two with DNA repair protein RAD23 and inhibitor of apoptosis protein. Four URGs were confirmed up-regulated by gamma irradiation by quantitative real-time PCR. One unknown gene, which was up-regulated to the greatest extent, might contribute to early recovery from gamma-irradiation-induced damage. The up-regulations of genes encoding DNA repair, protein processing, and energy metabolism proteins suggests that increases in gene products orchestrate DNA lesion repair and recover cellular functions in gamma-irradiated C. sinensis metacercariae.     

Molecular cloning and enzymatic characterization of a class mu glutathione <Emphasis Type="Italic">S</Emphasis>-transferase of <Emphasis Type="Italic">Paragonimus westermani</Emphasis>

Glutathione S-transferase (GST) is a component of a second line of defense against bioreactive radicals derived from host immune attack. Paragonimus westermani causes acute or chronic lung diseases in mammals. A cDNA clone, PwGST#11, of adult P. westermani produced in the present study was 748 bp long and encoded an open reading frame of 217 amino acids with a starting methionine. The molecular mass of this putative polypeptide, Pw26GST, was estimated to be 25.1 kDa with an isoelectric point of 5.7. Pw26GST was homologous with the 26-kDa GSTs of trematodes and vertebrates. Nine of the ten amino acid residues lining the glutathione-binding pocket were conserved. Putative Pw26GST polypeptide was clustered with 26-kDa GSTs of trematodes belonging to the class mu. Recombinant Pw26GST protein generated bacterially, revealed GST enzyme activity toward an universal and class mu-specific substrates. Mouse antisera to recombinant Pw26GST protein recognized native 26-kDa GST of P. westermani but not the GSTs of any other trematodes. Collectively, Pw26GST was found to be a member of class mu GSTs of P. westermani.     

Cofactor-independent phosphoglycerate mutase is an essential gene in procyclic form <Emphasis Type="Italic">Trypanosoma brucei</Emphasis>

Glycolysis and gluconeogenesis are, in part, driven by the interconversion of 3- and 2-phosphoglycerate (3-PG and 2-PG) which is performed by phosphoglycerate mutases (PGAMs) which can be cofactor dependant (dPGAM) or cofactor independent (iPGAM). The African trypanosome, Trypanosoma brucei, possesses the iPGAM form which is thought to play an important role in glycolysis. Here, we report on the use of RNA interference to down-regulate the T. brucei iPGAM in procyclic form T. brucei and evaluation of the resulting phenotype. We first demonstrated biochemically that depletion of the steady state levels of iPGM mRNA correlates with a marked reduction of enzyme activity. We further show that iPGAM is required for cell growth in procyclic T. brucei.     

Cytokine responses in mice infected with<Emphasis Type="Italic"> Clonorchis sinensis</Emphasis>

FVB and BALB/c mice show different morbidity, development of Clonorchis sinensis, and pathological changes following C. sinensis infection. FVB mice are susceptible and BALB/c mice are relatively more resistant to C. sinensis infection. To investigate the relationship between cytokine reaction and susceptibility to C. sinensis infection in FVB and BALB/c mice, we described both the patterns and kinetics of Th1 cytokines and Th2 cytokines in spleen cell culture. Interleukin (IL)-4 and IL-10 cytokine production in the culture supernatants of the concanavalin-A-stimulated spleen cells increased at 2–3 weeks post-infection in both strains. IL-5 production increased between 2 and 5 weeks post-infection in both strains, and reached a peak level at 2 weeks post-infection in BALB/c mice and 4 weeks post-infection in FVB mice. In contrast, gamma interferon (IFN-) production decreased between 2 and 4 weeks in both strains. IL-2 production increased slightly in BALB/c mice following infection, but was unchanged in FVB mice. IL-4 production over preinfection levels was significantly higher in FVB mice, whereas IFN-, IL-2, and IL-10 production were significantly higher in BALB/c mice. The levels of serum immunoglobulin E (IgE) and blood eosinophils in both mouse strains significantly increased between 3 and 6 weeks postinfection. Serum IgE levels were significantly higher in FVB mice than in BALB/c mice. The results of this study suggest that susceptibility to C. sinensis infection is associated with Th2 cytokine production, especially IL-4 which is predominant in relatively susceptible FVB mice.     

Lethal effect of ammonia on metacercariae of<Emphasis Type="Italic"> Clonorchis sinensis</Emphasis>

The effect of endogenous ammonia produced in dead fish or ammonia in ammonium bicarbonate solutions was evaluated on the viability of Clonorchis sinensis metacercariae. Viability was determined by worm recovery in rats infected with metacercariae exposed to ammonia in decaying fish or in vitro solutions. The recovery rates were 58%, 48%, 44% and 2% in fish that had been allowed to decay for 5, 10, 20 and 30 days at 4 degrees C, respectively, and this rate ( Y) was inversely correlated with the ammonia concentration ( X) in fish muscle extract ( Y=80.62-0.0667 X, r(2)=0.905, P=0.049). C. sinensis metacercariae barely survived in ammonia solutions with concentrations exceeding 1 g N/l. Our results indicate that the ammonia produced in dead fish is lethal to the viability of C. sinensis metacercariae.     

The <Emphasis Type="Italic">Wolbachia</Emphasis> endosymbiont of <Emphasis Type="Italic">Brugia malayi</Emphasis> has an active phosphoglycerate mutase: a candidate target for anti-filarial therapies

Phosphoglycerate mutases (PGM) interconvert 2- and 3-phosphoglycerate in the glycolytic and gluconeogenic pathways. A putative cofactor-independent phosphoglycerate mutase gene (iPGM) was identified in the genome sequence of the Wolbachia endosymbiont from the filarial nematode, Brugia malayi (wBm). Since iPGM has no sequence or structural similarity to the cofactor-dependent phosphoglycerate mutase (dPGM) found in mammals, it may represent an attractive Wolbachia drug target. In the present study, wBm–iPGM cloned and expressed in Escherichia coli was mostly insoluble and inactive. However, the protein was successfully produced in the yeast Kluyveromyces lactis and the purified recombinant wBm–iPGM showed typical PGM activity. Our results provide a foundation for further development of wBm–iPGM as a promising new drug target for novel anti-filarial therapies that selectively target the endosymbiont.     

Cysteine proteinases from promastigotes of <Emphasis Type="Italic">Leishmania</Emphasis> (<Emphasis Type="Italic">Viannia</Emphasis>) <Emphasis Type="Italic">braziliensis</Emphasis>

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.     

Cercarial shedding from <Emphasis Type="Italic">Galba</Emphasis><Emphasis Type="Italic">truncatula</Emphasis> infected with <Emphasis Type="Italic">Fasciola</Emphasis><Emphasis Type="Italic">gigantica</Emphasis> of distinct geographic origins

Galba truncatula snails were experimentally infected with either of two different isolates of Fasciola gigantica, originating from Egypt or China, to determine the influence of these isolates on the characteristics of snail infections. The survival rates of G. truncatula on day 30 post-exposure were 90.0% and 60.2% in the Egyptian and Chinese groups, respectively. The frequency of cercaria-shedding snails within the Egyptian group was 79.8%, whereas in the Chinese group it was 22.4%. The parasite origin had a significant effect on the durations of the prepatent and patent periods. The mean number of cercariae shed from the Egyptian group was significantly greater than that shed from the Chinese group (a mean of 275.5 per cercaria-shedding snail compared with 29.0). These results could be explained by the fact that G. truncatula might be a natural intermediate host for F. gigantica in Egypt, and the greater adaptability of the Egyptian miracidia of F. gigantica to unusual snail hosts. These results demonstrate the influence of the geographic origin of the parasite on the success of trematodes infecting snails.     

Differential gene expression profiling in human cholangiocarcinoma cells treated with <Emphasis Type="Italic">Clonorchis sinensis</Emphasis> excretory-secretory products

Clonorchiasis is associated with bile duct malignancy and the subsequent development of cholangiocarcinoma. Although this is likely caused by adult Clonorchis sinensis and its excretory–secretory products (ESP), the precise molecular mechanisms remain obscure. To evaluate the effect of C. sinensis infection on differential gene expression in host hepatocytes, we examined the kinetics of changes in gene expression in the human cholangiocarcinoma cell line HuCCT1 treated with ESP at different times. Using complementary DNA microarrays containing 23,920 human genes of known function, we initially identified 435 genes altered by ESP treatment. Of these, 31 were up-regulated and 35 were down-regulated more than twofold in a time-dependent manner following ESP treatment. Clustering of these genes by function revealed that several were involved in the cell cycle, oncogenesis, protein modification, immunity, signal transduction, cell structure, and developmental processes. These findings should provide a fundamental basis for further analysis of the molecular pathways and mechanisms involved in C. sinensis infection of host cells.