Optimized expression of Peptidyl-prolyl cis/transisomerase cyclophilinB with prokaryotic toxicity from Sporothrix globosa.

Cyclophilin B (CypB), a significant member of immunophilins family with peptidyl-prolyl cis-trans isomerase (PPIase) activity, is crucial for the growth and metabolism of prokaryotes and eukaryotes. Sporothrix globosa (S. globosa), a principal pathogen in the Sporothrix complex, causes sporotrichosis. Transcriptomic analysis identified the cypB gene as highly expressed in S. globosa. Our previous study demonstrated that the recombinant Escherichia coli strain containing SgcypB gene failed to produce sufficient product when it was induced to express the protein, implying the potential toxicity of recombinant protein to the bacterial host. Bioinformatics analysis revealed that SgCypB contains transmembrane peptides within the 52 amino acid residues at the N-terminus and 21 amino acids near the C-terminus, and 18 amino acid residues within the cytoplasm. AlphaFold2 predicted a SgCypB 3D structure in which there is an independent PPIase domain consisting of a spherical extracellular part. Hence, we chose to express the extracellular domain to yield high-level recombinant protein with PPIase activity. Finally, we successfully produced high-yield, truncated recombinant CypB protein from S. globosa (SgtrCypB) that retained characteristic PPIase activity without host bacterium toxicity. This study presents an alternative expression strategy for proteins toxic to prokaryotes, such as SgCypB. ONE-SENTENCE SUMMARY: The recombinant cyclophilin B protein of Sporothrix globosa was expressed successfully by retaining extracellular domain with peptidyl-prolyl cis-trans isomerase activity to avoid toxicity to the host bacterium.

Chitin-rich heteroglycan from Sporothrix schenckii sensu stricto potentiates fungal clearance in a mouse model of sporotrichosis and promotes macrophages phagocytosis.

BACKGROUND: Fungal cell wall polysaccharides maintain the integrity of fungi and interact with host immune cells. The immunomodulation of fungal polysaccharides has been demonstrated in previous studies. However, the effect of chitin-rich heteroglycan extracted from Sporothrix schenckii sensu stricto on the immune response has not been investigated. RESULTS: In this study, chitin-rich heteroglycan was extracted from S. schenckii sensu stricto, and immunomodulation was investigated via histopathological analysis of skin lesions in a mouse model of sporotrichosis and evaluation of the phagocytic function and cytokine secretion of macrophages in vitro. The results showed that the skin lesions regressed and granulomatous inflammation was reduced in infected mice within 5 weeks. Moreover, heteroglycan promoted the fungal phagocytosis by macrophages and modulated the cytokine secretion. Heteroglycan upregulated TNF-α expression early at 24 h and IL-12 expression late at 72 h after incubation, which might result from moderate activation of macrophages and contribute to the subsequent adaptive immune response. CONCLUSIONS: Chitin-rich heteroglycan extracted from S. schenckii sensu stricto potentiated fungal clearance in a mouse model of sporotrichosis. Moreover, chitin-rich heteroglycan promoted fungus phagocytosis by macrophages and modulated cytokines secretion. These results might indicate that chitin-rich heteroglycan could be considered as an immunomodulator used in the treatment of sporotrichosis.

Survival advantage depends on cecal volume rather than cecal length in a mouse model of cecal ligation and puncture.

BACKGROUND: Cecal ligation and puncture (CLP) is the most commonly used model to simulate human polymicrobial sepsis. However, the severity of CLP is difficult to be standardized across different laboratories. The aim of the present study was to evaluate the influence of ligated cecal volume and length on mortality in mouse CLP model. METHODS: Cecal length and volume were measured from 120 Kunming mice subjected to CLP or sham operation. According to cecal volume, mice were divided into three groups, volume0.0∼0.2 (0.0 cm(3)-0.2 cm(3)), volume0.2∼0.4 (0.2 cm(3)-0.4 cm(3)), and volume>0.4 (larger than 0.4 cm(3)). The contents of cytokines, including interleukin-1ß, interleukin-6, and TNF-α, were measured at 3 h after surgery. The blood bacterial load and oxidative stress indicators (including malondialdehyde and superoxide dismutase) were measured at 12 h after surgery. RESULTS: There was no significant difference on 72-h survival rate between the mice with cecum longer than 2 cm and shorter than 2 cm. Compared to the other volume groups, volume>0.4 group showed significantly increased blood bacterial load, malondialdehyde levels in lung and liver, and pro-inflammatory cytokines in serum. Surprisingly, the survival rate in volume>0.4 (0%) group showed significant difference from those of volume0.0∼0.2 group (40%) and volume0.2∼0.4 group (40%). CONCLUSIONS: The mice in volume>0.4 group have much serious inflammatory reaction and are easier to die. As the proportion of volume>0.4 mice is near 20%, it can have large influence on most of the related studies using this CLP model.

Should we abandon quinine plus antibiotic for treating uncomplicated falciparum malaria? A systematic review and meta-analysis of randomized controlled trials.

In this study, we compared the efficacies and adverse effects of quinine plus antibiotics and other anti-malaria drugs on treating uncomplicated falciparum malaria. By systematically searching the major databases PubMed, Embase, and the Cochrane Library, 14 randomized controlled trials (RCTs) including 1996 cases were identified. Then, we performed a systematic review and cumulative meta-analysis on these data. The primary outcome of these treatments was parasite failure at day 28. There was no significant difference between quinine plus antibiotic therapy (QACT) and artemisinin-based therapies (odds ratio (OR) 0.69, 95 % confidence interval (CI) 0.28 to 1.71) or non-artemisinin-based therapies except quinine monotherapy and chloroquine monotherapy (OR 0.56, 95 % CI 0.18 to 1.74). The secondary outcome was the adverse effects within 28 days, including nausea, dizziness, vomiting, diarrhea, abdominal pain, headache, and tinnitus. QACT significantly increased the risk of tinnitus compared with artemisinin-based therapies (OR 111.65, 95 % CI 12.63 to 986.87) and non-artemisinin-based therapies (OR 48.16, 95 % CI 16.23 to 142.92). Vomiting was more frequently reported in QACT compared with non-artemisinin-based therapies (OR 2.02, 95 % CI 1.14 to 3.56). This meta-analysis suggests that almost all regimens have equivalent treatment effect at the 28th day. However, the patients with QACT had a higher chance to suffer from vomiting and tinnitus. Therefore, QACT does not have significant advantage on treating uncomplicated falciparum malaria.

Antibodies against Clonorchis sinensis LDH could cross-react with LDHB localizing on the plasma membrane of human hepatocarcinoma cell SMMC-7721 and induce apoptosis.

Lactate dehydrogenase (LDH) is a terminal enzyme in anaerobic glycolytic pathway. It widely exists in various organisms and is in charge of converting the glycolysis product pyruvic acid to lactic acid. Most parasites, including Clonorchis sinensis, predominantly depend on glycolysis to provide energy. Bioinformatic analysis predicts that the LDHs from many species have more than one transmembrane region, suggesting that it may be a membrane protein. C. sinensis LDH (CsLDH) has been confirmed as a transmembrane protein mainly located in the tegument. The antibodies against CsLDH can inhibit the worm's energy metabolism, kill the worm, and may have the same effects on human cancer cells. In this study, we cloned and characterized human LDHA (HsLDHA), HsLDHB, and CsLDH. Semi-quantitative real-time RCP showed that HsLDHB only existed in hepatocarcinoma cell SMMC-7721. Confocal microscopy and Western blot experiments revealed that HsLDHB was localized in the plasma membrane of SMMC-7721 cells, and the antibodies against CsLDH could cross-react with it. This cross-reaction could inhibit the enzymatic activity of HsLDHB. The cancer cells co-cultured with anti-CsLDH sera showed a significant decrease in cell proliferation rate and increases in caspase 9 and reactive oxygen species (ROS) levels. Therefore, anti-CsLDH antibodies can induce the apoptosis of cancer cells SMMC-7721 and may serve as a new tool to inhibit tumor.

Analysis of global gene expression profiles suggests a role of acute inflammation in type 3C diabetes mellitus caused by pancreatic ductal adenocarcinoma.

AIMS/HYPOTHESIS: Pancreatic ductal adenocarcinoma (PDAC) can cause type 3C diabetes, known as PDAC-associated diabetes mellitus (PDAC-DM), but the mechanism is unknown. This study aimed to reveal the mechanism. METHODS: PDAC lesions from patients with or without PDAC-DM (n = 4 in each group) were individually profiled for 23,512 mRNAs with microarrays. Bioinformatic analysis and in vivo and in vitro assays were then conducted. RESULTS: We determined that 2,778 genes were differentially expressed; over-representation of ten genes was validated with quantitative RT-PCR. The analysis of gene ontology showed that the differentially expressed secretory genes were related mainly to inflammation. High levels of a marker of inflammation (C-reactive protein [CRP]) and an inflammatory mediator (TNF super-family member 13 [TNFSF13]) were found in the serum of patients with PDAC-DM. After surgical resection of PDAC lesions, CRP and TNFSF13 levels significantly decreased (p < 0.01). Furthermore, we found that the levels of TNFSF13 in PDAC lesions and TNFSF13 and CRP in serum were significantly correlated with the diabetic status of patients with PDAC-DM (p < 0.01). Assays in vivo showed that after exposure to an inhibitor of inflammation (celecoxib), the fasting blood glucose level in the mouse model of PDAC-DM dramatically decreased from 6.9 ± 0.1 to 5.6 ± 0.1 mmol/l in 2-4 days (p < 0.01). CONCLUSIONS/INTERPRETATION: We found that acute inflammation was involved in the pathogenesis of PDAC-DM. We contend that acute inflammation is a potential target for the diagnosis and treatment of PDAC-DM.

Genetic diversity of Plasmodium vivax population in Anhui province of China.

BACKGROUND: Although the numbers of malaria cases in China have been declining in recent years, outbreaks of Plasmodium vivax malaria were still being reported in rural areas south of the Yellow River. To better understand the transmission dynamics of P. vivax parasites in China, the extent of genetic diversity of P. vivax populations circulating in Bozhou of Anhui province of China were investigated using three polymorphic genetic markers: merozoite surface proteins 1 and 3α (pvmsp-1 and pvmsp-3α) and circumsporozoite protein (pvcsp). METHODS: Forty-five P. vivax clinical isolates from Bouzhou of Anhui province were collected from 2009 to 2010 and were analysed using PCR/RFLP or DNA sequencing. RESULTS: Seven and six distinct allelic variants were identified using PCR/RFLP analysis of pvmsp-3α with HhaI and AluI, respectively. DNA sequence analysis of pvmsp-1 (variable block 5) revealed that there were Sal-I and recombinant types but not Belem type, and seven distinct allelic variants in pvmsp-1 were detected, with recombinant subtype 2 (R2) being predominant (66.7%). All the isolates carried pvcsp with VK210 type but not VK247 or P. vivax-like types in the samples. Sequence analysis of pvcsp gene revealed 12 distinct allelic variants, with VK210-1 being predominant (41.5%). CONCLUSIONS: The present data indicate that there is some degree of genetic diversity among P. vivax populations in Anhui province of China. The genetic data obtained may assist in the surveillance of P. vivax infection in endemic areas or in tracking potential future disease outbreak.

Proteomic identification of potential Clonorchis sinensis excretory/secretory products capable of binding and activating human hepatic stellate cells.

Epidemiological and experimental evidence demonstrated that Clonorchis sinensis is an important risk factor of hepatic fibrosis and cholangiocarcinoma. C. sinensis excretory/secretory products (CsESPs) are protein complex including proteases, antioxidant enzymes, and metabolic enzymes, which may contribute to pathogenesis of liver fluke-associated hepatobiliary diseases. However, potential CsESP candidates involved into hepatic fibrosis and cholangiocarcinoma still remain to be elucidated. In the present study, we performed proteomic identification of CsESP candidates capable of binding and activating human hepatic stellate cell line LX-2. Immunofluorescence analysis confirmed the interaction of CsESPs with LX-2 cell membrane. LX-2 cells could be stimulated by CsESPs from 24 h post incubation (p < 0.05). Specifically, 50 µg/ml of CsESPs showed the strongest effect on cell proliferation in methyl thiazolyl tetrazolium (MTT) assay which could also be demonstrated by flow cytometry analysis (p < 0.01). Furthermore, expression level of human type III collagen in LX-2 cells treated with CsESPs was significantly higher than that in control cells measured by molecular beacon and semiquantitative reverse transcription (RT)-PCR approaches (p < 0.01). Finally, CsESPs before and after incubation with LX-2 cells were subjected to two-dimensional gel electrophoresis (2-DE) analysis and matrix associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. Nine proteins with abundance change above threefold were Rho GTPase-activating protein, mitochondrial cytochrome c oxidase subunit Va, α-enolase, phospholipase C, interleukin-15, insect-derived growth factor, cytochrome c oxidase subunit VI, DNAH1 protein, and kinesin light chain. Taken together, we identified potential CsESP candidates capable of binding and activating human hepatic stellate cells, providing more direct evidences that are previously unknown to accelerate strategies for C. sinensis prevention.

Schistosoma japonicum tegumental protein 20.8, role in reproduction through its calcium binding ability.

Schistosomiasis threatens thousands of millions of peoples' health every year in the world. Schistosoma japonicum, a pathogen of schistosomiasis, is covered by a lipid bilayer membrane which plays an important role in nutrient transport, signal transduction, interaction with host's immune system, etc. Thus, molecules in the tegumental membrane have gained more and more interest for understanding biological and pathological processes of schistosoma. In this study, we found a protein from S. japonicum cDNA library which has a 20.8 KDa molecular weight (SjTP20.8). Recombinant SjTP20.8 was produced and purified from Escherichia coli. The recombinant protein could be detected by S. japonicum-infected mice and human sera, and it had been found localizing in the tegumental membrane of S. japonicum in the section using immunofluorescence assay. In electrophoretic mobility shift assay, the protein could bind calcium iron in neutral condition. Result of cercariae challenge experiment indicates antibody against this protein can protect mice from chronic hepatic fibrosis. Our results indicate the S. japonicum tegumental protein 20.8 is crucial for the parasite's calcium absorbing and reproduction.

The serological diagnosis of human clonorchiasis by time-resolved fluoroimmunoassay based on GST2-specific IgG4 detection.

Due to its delayed fluorescence of a lanthanide chelate, high accuracy and low background the broad linear range, long fluorescent life-time and large Stoke's shift of europium chelates, the time-resolved fluorescence has been developed for higher sensitive immunoassay. In this article, a simple, sensitive and specific method-time-resolved fluoroimmunoassay (TRFIA) was adopted for immunoassay of clonorchiasis, and recombinant glutathione transferases 2 of Clonorchis sinensis (rCsGST2) was used as a diagnostic antigen. To evaluate this novel assay for clinical applications, 409 serum samples were investigated. The diagnostic accuracy of the antigen was evaluated by receiver-operating characteristic (ROC) analysis. The area under the ROC curve (AUC) was 0.965, 95% confidence interval (CI, 0.946, 0.985). To eliminate the random influence of ambient temperature, test parameters, photometric instruments and so on, the cut-off value was expressed as ratios between the fluorescence of sample and that of a well-defined negative control serum, and the deduced cut-off value was 9.3605. At the optimum cut-off criteria, the technique has a sensitivity of 95.80%, specificity of 93.60%. And the cross reactivity revealed that its cross reactivity with Schistosoma japonicum, round worm, hook worm, whip worm, and Toxoplasma gondii was 9.3, 8.3, 7.6, 9.8, and 5.0%, respectively. Kappa score of agreement between TRFIA and microscopic examination of stools was 0.892, P < 0.05. These combined results showed that our method is feasible and could be used for the clinical determination of clonorchiasis.

Biochemical characterization and functional analysis of fructose-1,6-bisphosphatase from Clonorchis sinensis.

Fructose-1,6-bisphosphatase (FBPase), a key regulatory enzyme of gluconeogenesis, plays an essential role in metabolism and development of most organisms. To the wealth of available knowledge about FBPase from Clonorchis sinensis (CsFBPase), in this study, the characteristics of CsFBPase and its potential role in pathogenesis of clonorchiasis were investigated. The Km value of CsFBPase was calculated to be 41.9 uM. The optimal temperature and pH of CsFBPase were 37 °C and pH 7.5-8.0, respectively. In addition, Mg(2+) or K(+) played a regulatory role in enzyme activity of CsFBPase. Both transcriptional and translational level of CsFBPase were higher in metacercariae (one of larva stages) than those in adult worm (P < 0.05). CsFBPase were observed to extensively express in the intestine, vitellaria and tegument of adult worms and ubiquitously in metacercariae. Moreover, CsFBPase was confirmed as a component of excretory/secretory products. Consequently, the translocation of CsFBPase could be detected on epithelial cells of bile duct in liver of C. sinensis infected rat. Recombinant CsFBPase can specifically bind to the membrane of human hepatic stellate cell line LX-2 by immunofluorescence analysis and stimulated proliferation and activation of LX-2 which demonstrated by Cell Counting Kit-8 and upregulation of key fibrosis-related factors, such as α-smooth muscle actin, collagen I and collagen III using qRT-PCR. Thus, we predicated that CsFBPase might be a multifunctional enzyme which played as both regulatory enzyme and virulence factor in pathogenesis of C. sinensis infection.

Expression, immunolocalization, and serological reactivity of a novel sphingomyelin phosphodiesterase-like protein, an excretory/secretory antigen from Clonorchis sinensis.

Clonorchiasis, caused by Clonorchis sinensis infection, is a zoonotic parasitic disease of hepatobiliary system in which the proteins released by adult are major pathogenetic factors. In this study, we first characterized a putative sphingomyelin phosphodiesterase (CsSMPase) A-like secretory protein, which was highly expressed in the adult worm. The full-length gene was cloned. The putative protein is of relatively low homology comparing with SMPase from other species, and of rich T cell and B cell epitopes, suggesting that it is an antigen of strong antigenicity. The complete coding sequence of the gene was expressed in the Escherichia coli. The recombinant CsSMPase (rCsSMPase) can be recognized by C. sinensis-infected serum, and the protein immunoserum can recognize a specific band in excretory/secretory products (ESPs) of C. sinensis adult by western blotting. Immunolocalization revealed that CsSMPase was not only localized on tegument, ventral sucker of metacercaria, and the intestine of adult but also on the nearby epithelium of bile duct of the infected Sprague-Dawley rats, implying that CsSMPase was mainly secreted and excreted through adult intestine and directly interacted with bile duct epithelium. Although immunized rats evoked high level antibody response, the antigen level was low in clonorchiasis patients. And the sensitivity and specificity of rCsSMPase were 50.0 % (12/24) and 88.4 % (61/69), in sera IgG-ELISA, respectively. It is likely due to the fact that CsSMPase binding to the plasma membrane of biliary epithelium decreases the antigen immune stimulation.

An antigenic recombinant serine protease from Trichinella spiralis induces protective immunity in BALB/c mice.

In this study, we report the cloning and characterization of a cDNA encoding a Trichinella serine protease gene (TspSP-1.3) from GenBank. The recombinant TspSP-1.3 protein (rTspSP-1.3) was expressed in an Escherichia coli expression system and purified with Ni-affinity chromatography. Real-time quantitative PCR analysis revealed that TspSP-1.3 was expressed at significantly higher levels in muscle larvae and adult worms than in newborn larvae. TspSP-1.3 was detected in excretory-secretory proteins of Trichinella spiralis with western blotting. Immunization with the rTspSP-1.3 antigen induced humoral immune responses, which manifested as elevated specific anti-rTspSP-1.3 IgG and IgE antibodies and a mixed Th1/Th2 response. To determine whether purified rTspSP-1.3 had good antigenicity and could be a vaccine candidate for the control of T. spiralis infection, we immunized BALB/c mice with rTspSP-1.3 and subsequently challenged the mice with T. spiralis larvae. The results showed that mice vaccinated with rTspSP-1.3 exhibited an average reduction in the muscle larvae burden of 39 % relative to the control group. These results suggest that TspSP-1.3 could be a novel vaccine candidate for controlling Trichinella infection.

Analysis of a novel cathepsin B circulating antigen and its response to drug treatment in Trichinella-infected mice.

In this paper, we cloned a novel full-length cDNA that encodes a Trichinella spiralis cathepsin B-like protease gene (TsCPB) using 3'-RACE PCR. The recombinant mature TsCPB protein (rTsCPB) was then expressed in an Escherichia coli expression system and purified with Ni-affinity chromatography. Real-time quantitative PCR revealed that TsCPB was expressed across all development stages of the parasite but had the highest expression level during the adult stage. Furthermore, rTsCPB was detected in Trichinella excretory-secretory products with anti-rTsCPB rabbit polyclonal antibodies. Interestingly, rTsCPB was strongly recognized by the T. spiralis-infected sera in Western blotting, implying that TsCPB protein appeared in the peripheral blood of Trichinella-infected mice as circulating antigens (CAg). We then analyzed the dynamic levels of TsCPB CAg and its antibodies in T. spiralis-infected sera by using an improved double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) and indirect ELISA, respectively. The results showed that TsCPB CAg can be detected much earlier compared to antibody detection in Trichinella-infected mice. In addition, we monitored the effects of albendazole drug therapy (a dosage of 370 mg/kg body weight, twice a day) on T. spiralis-infected mice by detecting the levels of TsCPB CAg and its antibody in the sera of drug-treated mice. The results showed that the levels of CAg dramatically decreased after successful drug treatment, while the antibody level remained unchanged. Overall, the novel Trichinella antigen TsCPB could be a promising novel circulating antigen molecule for the detection of Trichinella infection and for monitoring the efficacy of drug treatment of trichinellosis.

Identification and biochemical characterization of adenylate kinase 1 from Clonorchis sinensis.

Adenylate kinase 1 is responsible for the conversion of AMP into ADP involved in purine metabolism. In the present study, adenylate kinase 1 gene (CsADK1) was isolated from an adult cDNA library of Clonorchis sinensis, and the recombinant protein was expressed in Escherichia coli. Bioinformatics analysis implied that the putative protein contained 197 amino acids, and some residues in conservative binding sites of CsADK1 were substituted. The structure modeling analysis showed that CsADK1 was composed of a core domain, an NMP-binding domain, and a LID domain, which was just a small loop. It demonstrated that CsADK1 was a short isoform of ADKs. Moreover, CsADK1 was identified as an excretory/secretory product by western blot analysis. Real-time quantitative PCR showed that expression level of CsADK1 at the stage of excysted metacercaria was higher than those of adult worm (18.8-folds, P<0.01), metacercariae (1.5-folds, P<0.01), and eggs (5.6-folds, P<0.01). In addition, histochemistry analysis showed that CsADK1 was extensively distributed in metacercariae and in the vitellaria and eggs of adult worms. The Km and Vmax value for substrate ADP were 2.2 mM and 0.9 mM/min, respectively. The optimal temperature and pH value were 37 °C and from 7.5 to 8.0, respectively. The enzyme activity was highly dependent on Mg2+, and the optimal concentration of Mg2+ was 2 mM. However, the enzyme activity was slightly activated by Ca2+, and Mn2+ has no effect on activity. For monovalent ions, activity was highly activated by K+ and NH4+, but slightly by Li+. Taken together, CsADK1 was a metal ion-dependent enzyme involved in purine metabolism, which was important for development and reproduction, and might be a potential candidate for drug target for clonorchiasis.

Molecular identification and characterization of leucine aminopeptidase 2, an excretory-secretory product of Clonorchis sinensis.

Aminopeptidases serve vital roles in metabolism of hormones, neurotransmission, turnover of proteins and immunological regulations. Leucine aminopeptidases catalyze the hydrolysis of amino-acid residues from the N-terminus of proteins and peptides. In the present study, leucine aminopeptidase 2 (LAP2) gene of Clonorchis sinensis (C. sinensis) was isolated and identified from an adult cDNA library of C. sinensis. Recombinant CsLAP2 was expressed and purified in Escherichia coli BL21. The open reading frame of LAP2 contains 1,560 bp equivalent to 519 amino acids, a similarity analysis showed a relatively low homology with Homo sapiens (19.0 %), Trypanosoma cruzi (18.0 %), Mus musculus (19.3 %), and relatively high homology with Schistosoma mansoni (65.6 %). The optimum condition of rCsLAP2 enzyme activity was investigated using a fluorescent substrate of Leu-MCA at 37 °C and pH 7.5. The K (m) and V (max) values of rCsLAP2 were 18.2 µM and 10.7 µM/min, respectively. CsLAP2 gene expression can be detected at the stages of the adult worm, metacercaria, excysted metacercaria and egg of C. sinensis using real-time PCR, no difference was observed at the stages of the adult worm, metacercaria and egg. However, CsLAP2 showed a higher expression level at the stage of excysted metacercaria than the adult worm (3.90-fold), metacercaria (4.60-fold) and egg (4.59-fold). Histochemistry analysis showed that CsLAP2 was located at the tegument and excretory vesicle of metacercaria, and the tegument and intestine of adult worm. The immune response specific to rCsLAP2 was characterized by a mixed response patterns of Th1 and Th2, indicating a compounded humoral and cellular immune response. The combined results from the present study indicate that CsLAP2 was an important antigen exposed to host immune system, and probably implicated as potential role in interaction with host cells in clonorchiasis.

Serological diagnosis of clonorchiasis: using a recombinant propeptide of cathepsin L proteinase from Clonorchis sinensis as a candidate antigen.

Clonorchiasis is a common zoonosis in southern and northeastern parts of China, especially in Guangdong, Guangxi and Jilin province. Anti-Clonorchis sinensis antibody detection by enzyme-linked immunosorbent assay (ELISA) has been used for epidemiological surveys of clonorchiasis for its convenience and celerity, but it is still a meaningful work to screen ideal diagnostic antigen or antibody subtype for improvement of diagnostic sensitivity and specificity and for judgement of curative effect. In the present study, recombinant CsCatL-propeptide (rCsCatL-propeptide) was highly expressed in form of inclusion body in Escherichia coli. Soluble rCsCatL-propeptide with high purity were obtained after purification in denatured condition by using His Bind Purification kit, and then renatured. The major antibody subtypes responding to rCsCatL-propeptide in sera from clonorchiasis patients were IgG1 and IgG4, but the level of IgG4 was more predominant (P < 0.05). The sensitivity of specific IgG4 detection (91.7%) was statistically significantly higher than that of IgG1 (25.0%) with rCsCatL-propeptide (P < 0.01). The specificities of IgG1 and IgG4 detection with rCsCatL-propeptide were 83.3% and 88.5%, respectively, and the difference between them was not statistically significant (P > 0.05). Cross-reactions took place when we detected IgG1 of sera from patients infected with Schistosoma japonicum, Paragonimus westermani, hookworm, Trichuris trichiura and Ascaris lumbricoides with rCsCatL-propeptide, while cross-reactions only took place in sera from patients infected with S. japonicum and P. westermani when we detected specific IgG4. The positive rate of IgG4 detection in sera from clonorchiasis patients with <1,000, 1,000-4,999, 5,000-9,999, and ≥10,000 eggs per gram faeces (EPG) were 76.9%, 89.3%, 95.6%, and 100.0%, respectively. The positive rates of serodiagnosis correlated well with the EPG (r = 0.93). Overall, rCsCatL-propeptide is a valuable candidate for specific IgG4 detection in sera from clonorchiasis patients by the method of ELISA for its few cross-reaction and acceptable sensitivity. In addition, specific IgG4 detection can be used to valuate infected degree and therapeutic effect of clonorchiasis patients.

Gene cloning, expression, and localization of antigen 5 in the life cycle of Echinococcus granulosus.

Antigen 5 (Ag5) has been identified as a dominant component of cyst fluid of Echinococcus granulosus and is considered as a member of serine proteases family, which in other helminth, plays an important role in the egg hatch and larva invasion. However, whether Ag5 is expressed and secreted in all life stages is unknown. In this study, according to the sequence in GenBank, we cloned and sequenced the open reading frame (ORF) of Ag5 gene from the protoscolices of E. granulosus isolated from the sheep in Qinhai Province of China, and found several substitutions and a base insert and deletion in a short region near the stop code, leading to a frameshift mutation which is conserved with the homologue of other cestode. The ORF is 1,455 bp in length, encoding 484 amino acids with a secretory signal peptide. Bioinformatics analysis predicted several phosphorylation and myristoylation sites and a N-glycosylation site and a species-specific linear B epitope in the protein. The ORF was cloned into the plasmid pET28a(+) vector and expressed in Escherichia coli . The recombinant protein was purified by affinity chromatography. Anti-rEgAg5 antiserum was prepared in rats and used to analyze the localization of Ag5 in protoscolex and adult worm by immunofluorescence technique. Results demonstrated that the Ag5 is strongly expressed in the tegument of protoscolex and the embryonic membrane of egg and surface of oncosphere; meanwhile, it is also weakly expressed in tegument of the adult. This study showed that Ag5 is expressed in all stages of life cycle, secreted from the surface of the worm and may be anchored in membrane by its myristoylation sites; these characteristics make it a candidate antigen for diagnosis and vaccine for both intermediate and definitive hosts.

41.5-kDa Cathepsin L protease from Clonorchis sinensis: expression, characterization, and serological reactivity of one excretory-secretory antigen.

Cysteine proteases (CPs) were associated with the pathogenicity and excystment of Clonorchis sinensis. Most of them were potential antigens for the immunodiagnosis of clonorchiasis. More researches on CPs will let us know more about their functions, and further employ them for the development of more efficient diagnostic reagent and prevention strategies. In the current study, a full-length sequence encoding cathepsin L from C. sinensis (CsCL41.5) was identified from our adult cDNA library. Bioinformatic analysis showed that CsCL41.5 included typical motifs of cathepsin L (ERFNIN and GNFD motifs) and conserved amino acid positions which constituted the active center of the enzyme. The identity of its amino acid sequence with the cathepsin L of Schistosoma japonicum was 49.6 %. Recombinant CsCL41.5 (rCsCL41.5) was highly expressed in the form of inclusion body in Escherichia coli, and soluble rCsCL41.5 was obtained after purification and renaturation. Western blotting analysis indicated that CsCL41.5 is an excretory-secretory antigen of C. sinensis adult. Immunolocalization demonstrated that CsCL41.5 is distributed in the intestine and eggs in the uterus of adult worm, tegument of metacercaria, oral suck, and tail of cercaria. ELISA assays showed that IgG4 was the predominant IgG isotype responding to rCsCL41.5 in sera from clonorchiasis patients. The sensitivity and specificity of specific IgG4 detection with rCsCL41.5 was 62.5 % (15/24) and 81.7 % (49/60), respectively. It was concluded that there were differences in biological function, efficiency of serodiagnosis, and characterization of immune reactivity between CsCL41.5 and other CPs of C. sinensis, combining with previous studies.

Molecular expression and characterization of a novel protein phosphatase 2A gene from Clonorchis sinensis.

Reversible phosphorylation of proteins is a critical mechanism involved in physiological function of organisms, including Clonorchis sinensis. In the present study, One cDNA clone encoding protein phosphatase 2A (CsPP2A) was isolated from a C. sinensis adult cDNA plasmid library. The open reading frame of the novel gene contains 924 bp and encoded a putative protein of 307 amino acids. A similarity analysis showed high homology with Schistosoma japonicum (76.3%) and Homo sapiens (84.4%), respectively. Recombinant CsPP2A (rCsPP2A) was expressed and purified from Escherichia coli BL21 using pET28a (+) as an expression vector. CsPP2A showed higher transcript level in adult worm but excysted metacercaria (P > 0.05), metacercaria (P < 0.05), and egg (P < 0.05) using real-time RT-PCR. Western blotting analysis showed that rCsPP2A could be identified by anti-rCsPP2A rat serum, C. sinensis-infected rat serum, and the serum from the rats immunized with excretory-secretory products of C. sinensis. Immunohistochemical assay showed that CsPP2A was deposited at the egg, the vitellarium of adult worm, and the excretory bladder of metacercaria. Collectively, the results of this study suggested that CsPP2A may be involved in the development of adult and metacercaria of C. sinensis.