Prostaglandin synthase activity of sigma- and mu-class glutathione transferases in a parasitic trematode, Clonorchis sinensis.

Sigma-class glutathione transferase (GST) proteins with dual GST and prostaglandin synthase (PGS) activities play a crucial role in the establishment of Clonorchis sinensis infection. Herein, we analyzed the structural and enzymatic properties of sigma-class GST (CsGST-σ) proteins to obtain insight into their antioxidant and immunomodulatory functions in comparison with mu-class GST (CsGST-µ) proteins. CsGST-σ proteins conserved characteristic structures, which had been described in mammalian hematopoietic prostaglandin D2 synthases. Recombinant forms of these CsGST-σ and CsGST-µ proteins expressed in Escherichia coli exhibited considerable degrees of GST and PGS activities with substantially different specific activities. All recombinant proteins displayed higher affinities toward prostaglandin H2 (PGS substrate; average Km of 30.7 and 3.0 µm for prostaglandin D2 [PGDS] and E2 synthase [PGES], respectively) than those toward CDNB (GST substrate; average Km of 1,205.1 µm). Furthermore, the catalytic efficiency (Kcat/Km) of the PGDS/PGES activity was higher than that of GST activity (average Kcat/Km of 3.1, 0.7, and 7.0×10-3 s-1µm-1 for PGDS, PGES, and GST, respectively). Our data strongly suggest that the C. sinensis sigma- and mu-class GST proteins are deeply involved in regulating host immune responses by generating PGD2 and PGE2 in addition to their roles in general detoxification.

Genome-wide identification of histone lysine methyltransferases and their implications in the epigenetic regulation of eggshell formation-related genes in a trematode parasite Clonorchis sinensis.

Epigenetic writers including DNA and histone lysine methyltransferases (DNMT and HKMT, respectively) play an initiative role in the differentiation and development of eukaryotic organisms through the spatiotemporal regulation of functional gene expressions. However, the epigenetic mechanisms have long been suspected in helminth parasites lacking the major DNA methyltransferases DNMT1 and DNMT3a/3b. Very little information on the evolutionary status of the epigenetic tools and their role in regulating chromosomal genes is currently available in the parasitic trematodes. We previously suggested the probable role of a DNMT2-like protein (CsDNMT2) as a genuine epigenetic writer in a trematode parasite Clonorchis sinensis. Here, we analyzed the phylogeny of HKMT subfamily members in the liver fluke and other platyhelminth species. The platyhelminth genomes examined conserved genes for the most of SET domain-containing HKMT and Disruptor of Telomeric Silencing 1 subfamilies, while some genes were expanded specifically in certain platyhelminth genomes. Related to the high gene dosages for HKMT activities covering differential but somewhat overlapping substrate specificities, variously methylated histones were recognized throughout the tissues/organs of C. sinensis adults. The temporal expressions of genes involved in eggshell formation were gradually decreased to their lowest levels proportionally to aging, whereas those of some epigenetic tool genes were re-boosted in the later adult stages of the parasite. Furthermore, these expression levels were significantly affected by treatment with DNMT and HKMT inhibitors. Our data strongly suggest that methylated histones are potent epigenetic markers that modulate the spatiotemporal expressions of C. sinensis genes, especially those involved in sexual reproduction.

Hypoxic and nitrosative stress conditions modulate expression of myoglobin genes in a carcinogenic hepatobiliary trematode, Clonorchis sinensis.

Despite recent evidence suggesting that adult trematodes require oxygen for the generation of bioenergy and eggshells, information on the molecular mechanism by which the parasites acquire oxygen remains largely elusive. In this study, the structural and expressional features of globin genes identified in Clonorchis sinensis, a carcinogenic trematode parasite that invades the hypoxic biliary tracts of mammalian hosts, were investigated to gain insight into the molecules that enable oxygen metabolism. The number of globin paralogs substantially differed among parasitic platyhelminths, ranging from one to five genes, and the C. sinensis genome encoded at least five globin genes. The expression of these Clonorchis genes, named CsMb (CsMb1-CsMb3), CsNgb, and CsGbX, according to their preferential similarity patterns toward respective globin subfamilies, exponentially increased in the worms coinciding with their sexual maturation, after being downregulated in early juveniles compared to those in metacercariae. The CsMb1 protein was detected throughout the parenchymal region of adult worms as well as in excretory-secretory products, whereas the other proteins were localized exclusively in the sexual organs and intrauterine eggs. Stimuli generated by exogenous oxygen, nitric oxide (NO), and nitrite as well as co-incubation with human cholangiocytes variously affected globin gene expression in live C. sinensis adults. Together with the specific histological distributions, these hypoxia-induced patterns may suggest that oxygen molecules transported by CsMb1 from host environments are provided to cells in the parenchyma and intrauterine eggs/sex organs of the worms for energy metabolism and/or, more importantly, eggshell formation by CsMb1 and CsMb3, respectively. Other globin homologs are likely to perform non-respiratory functions. Based on the responsive expression profile against nitrosative stress, an oxygenated form of secreted CsMb1 is suggested to play a pivotal role in parasite survival by scavenging NO generated by host immune cells via its NO dioxygenase activity.

Corrigendum: Eosinophil Activation by Toll-Like Receptor 4 Ligands Regulates Macrophage Polarization.

[This corrects the article DOI: 10.3389/fcell.2019.00329.].

Eosinophil Activation by Toll-Like Receptor 4 Ligands Regulates Macrophage Polarization.

Eosinophils are terminally differentiated granulocytes that have long been considered as destructive cells associated with Th2 type immune responses such as allergic inflammation and helminth infections. Recently, eosinophils have been actively studied as multifunctional leukocytes regulating an array of physiological responses through interaction with other immune cells. In this study, we examined the expression and function of Toll-like receptors (TLRs) in eosinophilic EoL-1 cells and demonstrated the expression of a number of immune mediators in activated EoL-1 cells and their interaction with the macrophage cell line THP-1 upon TLR4 ligand stimulation. EoL-1 cells differentiated with butyrate increased expression of TLR3, TLR4, and TLR7 at mRNA and protein level with flow cytometry analysis. Mature eosinophils derived from human cord blood CD34+ cells were subjected to RNA-sequencing, and showed the expression of a panel of TLR transcripts and TLR4 was the most highly expressed TLR. Among the cognate ligands of TLR3, TLR4, and TLR7, lipopolysaccharide (LPS) or palmitic acid significantly increased mRNA expression of immune mediators in differentiated EoL-1 cells. Notably, Western blot analysis of palmitic acid-treated differentiated EoL-1 cells showed significantly up-regulated expression of Th2 type cytokines and transcription factors driving eosinophil differentiation. To evaluate functional significance of TLR4 ligand-stimulated eosinophils, we added conditioned media (CM) from EoL-1 cells to differentiated THP-1 cells and assessed the expression of M1 macrophage or M2 macrophage-related markers. M1 and M2 macrophage markers were significantly upregulated by CM from LPS and palmitic acid stimulated EoL-1 cells, respectively. In addition, the adipose tissue of obese mice, where eosinophils are decreased due to obesity-induced inflammation, showed significantly decreased frequency of M2 macrophages, despite an increase in the total macrophage numbers. Based on these collective data, we proposed that eosinophils regulate both inflammatory and anti-inflammatory polarization of macrophages through functional changes induced by different TLR4 ligands.

Molecular characteristics and induction profiles of hypoxia-inducible factor-1α and other basic helix-loop-helix and Per-Arnt-Sim domain-containing proteins identified in a carcinogenic liver fluke Clonorchis sinensis.

Clonorchis sinensis (C. sinensis), a trematode parasite that invades the hypoxic hepatobiliary tract of vertebrate hosts requires a considerable amount of oxygen for its sexual reproduction and energy metabolism. However, little is known regarding the molecular mechanism of C. sinensis involved in the adaptation to the hypoxic environments. In this study, we investigated the molecular structures and induction patterns of hypoxia-inducible factor-1α (HIF-1α) and other basic helix-loop-helix and Per-Arnt-Sim (bHLH-PAS) domain-containing proteins such as HIF-1ß, single-minded protein and aryl hydrocarbon receptor, which might prompt adaptive response to hypoxia, in C. sinensis. These proteins possessed various bHLH-PAS family-specific domains. Expression of C. sinensis HIF-1α (CsHIF-1α) was highly induced in worms which were either exposed to a hypoxic condition or co-incubated with human cholangiocytes. In addition to oxygen, nitric oxide and nitrite affected the CsHIF-1α expression depending on the surrounding oxygen concentration. Treatment using a prolyl hydroxylase-domain protein inhibitor under 20%-oxygen condition resulted in an increase in the CsHIF-1α level. Conversely, the other bHLH-PAS genes were less responsive to these exogenous stimuli. We suggest that nitrite and nitric oxide, as well as oxygen, coordinately involve in the regulation of HIF-1α expression to adapt to the hypoxic host environments in C. sinensis.

Fasciclin-calcareous corpuscle binary complex mediated protein-protein interactions in Taenia solium metacestode.

BACKGROUND: Neurocysticercosis (NC) caused by Taenia solium metacestode (TsM) is a serious neurological disease of global concern. Diverse bioactive molecules involved in the long-term survival of TsM might contribute to disease progression. Fasciclin (Fas) is an extracellular protein that mediates adhesion, migration and differentiation of cells by interacting with other molecules. We hypothesized that TsMFas might bind to calcareous corpuscle (CC) through its adhesive property and participate in crucial protein-protein interactions, thus contributing to the creation of a symbiotic interactome network. METHODS: Two paralogous TsMFas (TsMFas1 and TsMFas2) were isolated, and their molecular properties were characterized. The co-localization pattern of TsMFas1 and TsMFas2 with CC was determined. CC-TsMFas binary complex was generated by incubating CC with recombinant proteins (rTsMFas1 and 2). In vitro binding assay of CC-rTsMFas1 or CC-rTsMFas2 binary complex with TsM cellular proteins extracted from scolex and neck was conducted. Their binding partners were identified through proteomic analysis. Integrated protein-protein interaction networks were established. RESULTS: TsMFas1 (6072 bp long) was composed of 15 exons (841 amino acid polypeptide) interrupted by 14 introns. TsMFas2 (5201 bp long) comprised of 11 exons (597 amino acids) and 10 intervening introns. These proteins displayed 22% amino acid sequence identity to each other, but tightly conserved Fas-related domains. Several isoforms of Fas1 and Fas2 proteins might have been expressed through post-translational modifications. They showed adhesion activity with other cells. TsMFas proteins were largely distributed in parenchymal regions of the scolex and bladder wall. These molecules were co-localized with CC, a unique organelle found in platyhelminths. Subsequent proteome analysis of CC-Fas binary complex mediated protein-protein interactions revealed seven protein ligands in the TsM cellular proteins. Their functions were mainly segregated into carbohydrate metabolism (enolase, phosphoenolpyruvate carboxykinase, phosphoglycerate kinase and glyceraldehyde 3-phosphate dehydrogenase) and cytoskeleton/cellular motility (actin, paramyosin and innexin nuc-9). Those proteins had direct (physical) and/or indirect (functional) relationships along with their biochemical properties and biological roles. CONCLUSION: Protein repertoires strongly suggest that TsMFas and CC may symbiotically mediate protein-protein interactions during biological processes to maintain efficacious homeostatic functions and ensure the prolonged survival of TsM in the host.

Isolation and Characterization of Vaccine Candidate Genes Including CSP and MSP1 in Plasmodium yoelii.

Malaria is an infectious disease affecting humans, which is transmitted by the bite of Anopheles mosquitoes harboring sporozoites of parasitic protozoans belonging to the genus Plasmodium. Despite past achievements to control the protozoan disease, malaria still remains a significant health threat up to now. In this study, we cloned and characterized the full-unit Plasmodium yoelii genes encoding merozoite surface protein 1 (MSP1), circumsporozoite protein (CSP), and Duffy-binding protein (DBP), each of which can be applied for investigations to obtain potent protective vaccines in the rodent malaria model, due to their specific expression patterns during the parasite life cycle. Recombinant fragments corresponding to the middle and C-terminal regions of PyMSP1 and PyCSP, respectively, displayed strong reactivity against P. yoelii-infected mice sera. Specific native antigens invoking strong humoral immune response during the primary and secondary infections of P. yoelii were also abundantly detected in experimental ICR mice. The low or negligible parasitemia observed in the secondary infected mice was likely to result from the neutralizing action of the protective antibodies. Identification of these antigenic proteins might provide the necessary information and means to characterize additional vaccine candidate antigens, selected solely on their ability to produce the protective antibodies.

Lineage-specific expansion and loss of tyrosinase genes across platyhelminths and their induction profiles in the carcinogenic oriental liver fluke, Clonorchis sinensis.

Tyrosinase provides an essential activity during egg production in diverse platyhelminths by mediating sclerotization of eggshells. In this study, we investigated the genomic and evolutionary features of tyrosinases in parasitic platyhelminths whose genomic information is available. A pair of paralogous tyrosinases was detected in most trematodes, whereas they were lost in cyclophyllidean cestodes. A pseudophyllidean cestode displaying egg biology similar to that of trematodes possessed an orthologous gene. Interestingly, one of the paralogous tyrosinases appeared to have been multiplied into three copies in Clonorchis sinensis and Opisthorchis viverrini. In addition, a fifth tyrosinase gene that was minimally transcribed through all developmental stages was further detected in these opisthorchiid genomes. Phylogenetic analyses demonstrated that the tyrosinase gene has undergone duplication at least three times in platyhelminths. The additional opisthorchiid gene arose from the first duplication. A paralogous copy generated from these gene duplications, except for the last one, seemed to be lost in the major neodermatans lineages. In C. sinensis, tyrosinase gene expressions were initiated following sexual maturation and the levels were significantly enhanced by the presence of O2 and bile. Taken together, our data suggest that tyrosinase has evolved lineage-specifically across platyhelminths related to its copy number and induction mechanism.

Codon Usage Patterns of Tyrosinase Genes in Clonorchis sinensis.

Codon usage bias (CUB) is a unique property of genomes and has contributed to the better understanding of the molecular features and the evolution processes of particular gene. In this study, genetic indices associated with CUB, including relative synonymous codon usage and effective numbers of codons, as well as the nucleotide composition, were investigated in the Clonorchis sinensis tyrosinase genes and their platyhelminth orthologs, which play an important role in the eggshell formation. The relative synonymous codon usage patterns substantially differed among tyrosinase genes examined. In a neutrality analysis, the correlation between GC12 and GC3 was statistically significant, and the regression line had a relatively gradual slope (0.218). NC-plot, i.e., GC3 vs effective number of codons (ENC), showed that most of the tyrosinase genes were below the expected curve. The codon adaptation index (CAI) values of the platyhelminth tyrosinases had a narrow distribution between 0.685/0.714 and 0.797/0.837, and were negatively correlated with their ENC. Taken together, these results suggested that CUB in the tyrosinase genes seemed to be basically governed by selection pressures rather than mutational bias, although the latter factor provided an additional force in shaping CUB of the C. sinensis and Opisthorchis viverrini genes. It was also apparent that the equilibrium point between selection pressure and mutational bias is much more inclined to selection pressure in highly expressed C. sinensis genes, than in poorly expressed genes.

Recurrent emergence of structural variants of LTR retrotransposon CsRn1 evolving novel expression strategy and their selective expansion in a carcinogenic liver fluke, Clonorchis sinensis.

Autonomous retrotransposons, in which replication and transcription are coupled, encode the essential gag and pol genes as a fusion or separate overlapping form(s) that are expressed in single transcripts regulated by a common upstream promoter. The element-specific expression strategies have driven development of relevant translational recoding mechanisms including ribosomal frameshifting to satisfy the protein stoichiometry critical for the assembly of infectious virus-like particles. Retrotransposons with different recoding strategies exhibit a mosaic distribution pattern across the diverse families of reverse transcribing elements, even though their respective distributions are substantially skewed towards certain family groups. However, only a few investigations to date have focused on the emergence of retrotransposons evolving novel expression strategy and causal genetic drivers of the structural variants. In this study, the bulk of genomic and transcribed sequences of a Ty3/gypsy-like CsRn1 retrotransposon in Clonorchis sinensis were analyzed for the comprehensive examination of its expression strategy. Our results demonstrated that structural variants with single open reading frame (ORF) have recurrently emerged from precedential CsRn1 copies encoding overlapping gag-pol ORFs by a single-nucleotide insertion in an upstream region of gag stop codon. In the parasite genome, some of the newly evolved variants appeared to undergo proliferative burst as active master lineages together with their ancestral copies. The genetic event was similarly observed in Opisthorchis viverrini, the closest neighbor of C. sinensis, whereas the resulting structural variants might have failed to overcome purifying selection and comprised minor remnant copies in the Opisthorchis genome.

Biochemical Characterization of Echinococcus multilocularis Antigen B3 Reveals Insight into Adaptation and Maintenance of Parasitic Homeostasis at the Host-Parasite Interface.

Alveolar echinococcosis (AE) caused by Echinococcus multilocularis metacestode is frequently associated with deleterious zoonotic helminthiasis. The growth patterns and morphological features of AE, such as invasion of the liver parenchyme and multiplication into multivesiculated masses, are similar to those of malignant tumors. AE has been increasingly detected in several regions of Europe, North America, Central Asia, and northwestern China. An isoform of E. multilocularis antigen B3 (EmAgB3) shows a specific immunoreactivity against patient sera of active-stage AE, suggesting that EmAgB3 might play important roles during adaptation of the parasite to hosts. However, expression patterns and biochemical properties of EmAgB3 remained elusive. The protein profile and nature of component proteins of E. multilocularis hydatid fluid (EmHF) have never been addressed. In this study, we conducted proteome analysis of EmHF of AE cysts harvested from immunocompetent mice. We observed the molecular and biochemical properties of EmAgB3, including differential transcription patterns of paralogous genes, macromolecular protein status by self-assembly, distinct oligomeric states according to individual anatomical compartments of the worm, and hydrophobic ligand-binding protein activity. We also demonstrated tissue expression patterns of EmAgB3 transcript and protein. EmAgB3 might participate in immune response and recruitment of essential host lipids at the host-parasite interface. Our results might contribute to an in depth understanding of the biophysical and biological features of EmAgB3, thus providing insights into the design of novel targets to control AE.

Spatiotemporal Expression Patterns and Antibody Reactivity of Taeniidae Endophilin B1.

Larval Taeniidae, such as metacestodes of Taenia solium, Echinococcus granulosus, and Echinococcus multilocularis, produce chronic and fatal helminthic diseases. Proper identification of these zoonotic cestodiases is often challenging and is hampered in some clinical settings. Endophilin B1 plays critical roles in the maintenance of membrane contours and endocytosis. We isolated proteins homologous to endophilin B1 from T. solium, Taenia saginata, and Taenia asiatica The three Taeniidae endophilin B1 proteins shared 92.9 to 96.6% sequence identity. They harbored a Bin1/amphiphysin/Rvs (BAR) domain and residues for a dimeric interface but lacked a SRC homology 3 (SH3) domain. Endophilin B1 showed a unique immunological profile and was abundantly expressed in the tegumental syncytium of Taeniidae metacestodes and adults. Bacterially expressed recombinant T. solium endophilin B1 (rTsMEndoB1) demonstrated a sensitivity of 79.7% (345/433 cases) for serodiagnosis of larval Taeniidae infections. The protein showed strong immune recognition patterns against sera from patients with chronic neurocysticercosis, cystic echinococcosis, or advanced-stage alveolar echinococcosis. Adult Taeniidae infections exhibited moderate degrees of positive antibody responses (65.7% [23/35 samples]). rTsMEndoB1 showed some cross-reactivity with sera from patients infected with Diphyllobothriidae (23.6% [25/106 samples]) but not with sera from patients with other parasitic diseases or normal controls. The specificity was 91.7% (256/301 samples). The positive and negative predictive values were 93.6% and 73.4%, respectively. Our results demonstrate that Taeniidae endophilin B1 may be involved in the control of membrane dynamics, thus contributing to shaping and maintaining the tegumental curvature. rTsMEndoB1 may be useful for large-scale screening, as well as for individual diagnosis and follow-up surveillance of Taeniidae infections.

Evolutionary characterization of Ty3/gypsy-like LTR retrotransposons in the parasitic cestode Echinococcus granulosus.

Cyclophyllidean cestodes including Echinococcus granulosus have a smaller genome and show characteristics such as loss of the gut, a segmented body plan, and accelerated growth rate in hosts compared with other tissue-invading helminths. In an effort to address the molecular mechanism relevant to genome shrinkage, the evolutionary status of long-terminal-repeat (LTR) retrotransposons, which are known as the most potent genomic modulators, was investigated in the E. granulosus draft genome. A majority of the E. granulosus LTR retrotransposons were classified into a novel characteristic clade, named Saci-2, of the Ty3/gypsy family, while the remaining elements belonged to the CsRn1 clade of identical family. Their nucleotide sequences were heavily corrupted by frequent base substitutions and segmental losses. The ceased mobile activity of the major retrotransposons and the following intrinsic DNA loss in their inactive progenies might have contributed to decrease in genome size. Apart from the degenerate copies, a gag gene originating from a CsRn1-like element exhibited substantial evidences suggesting its domestication including a preserved coding profile and transcriptional activity, the presence of syntenic orthologues in cestodes, and selective pressure acting on the gene. To my knowledge, the endogenized gag gene is reported for the first time in invertebrates, though its biological function remains elusive.

Activating transcription factor-3 induction is involved in the anti-inflammatory action of berberine in RAW264.7 murine macrophages.

Berberine is an isoquinoline alkaloid found in Rhizoma coptidis, and elicits anti-inflammatory effects through diverse mechanisms. Based on previous reports that activating transcription factor-3 (ATF-3) acts as a negative regulator of LPS signaling, the authors investigated the possible involvement of ATF-3 in the anti-inflammatory effects of berberine. It was found berberine concentration-dependently induced the expressions of ATF-3 at the mRNA and protein levels and concomitantly suppressed the LPS-induced productions of proinflammatory cytokines (TNF-α, IL-6, and IL-1ß). In addition, ATF-3 knockdown abolished the inhibitory effects of berberine on LPS-induced proinflammatory cytokine production, and prevented the berberine-induced suppression of MAPK phosphorylation, but had little effect on AMPK phosphorylation. On the other hand, the effects of berberine, that is, ATF-3 induction, proinflammatory cytokine inhibition, and MAPK inactivation, were prevented by AMPK knockdown, suggesting ATF-3 induction occurs downstream of AMPK activation. The in vivo administration of berberine to mice with LPS-induced endotoxemia increased ATF-3 expression and AMPK phosphorylation in spleen and lung tissues, and concomitantly reduced the plasma and tissue levels of proinflammatory cytokines. These results suggest berberine has an anti-inflammatory effect on macrophages and that this effect is attributable, at least in part, to pathways involving AMPK activation and ATF-3 induction.

Clonorchis sinensis omega-class glutathione transferases play major roles in the protection of the reproductive system during maturation and the response to oxidative stress.

BACKGROUND: Clonorchis sinensis causes a major food-borne helminthic infection. This species locates in mammalian hepatobiliary ducts, where oxidative stressors and hydrophobic substances are profuse. To adapt to the hostile micromilieu and to ensure its long-term survival, the parasite continuously produces a diverse repertoire of antioxidant enzymes including several species of glutathione transferases (GSTs). Helminth GSTs play pertinent roles during sequestration of harmful xenobiotics since most helminths lack the cytochrome P-450 detoxifying enzyme. METHODS: We isolated and analyzed the biochemical properties of two omega-class GSTs of C. sinensis (CsGSTo1 and CsGSTo2). We observed spatiotemporal expression patterns in accordance with the maturation of the worm's reproductive system. Possible biological protective roles of CsGSTos in these organs under oxidative stress were investigated. RESULTS: The full-length cDNAs of CsGSTo1 and 2 constituted 965 bp and 1,061 bp with open reading frames of 737 bp (246 amino acids) and 669 bp (223 amino acids). They harbored characteristic N-terminal thioredoxin-like and C-terminal α-helical domains. A cysteine residue, which constituted omega-class specific active site, and the glutathione-binding amino acids, were recognized in appropriate positions. They shared 44 % sequence identity with each other and 14.8-44.8 % with orthologues/homologues from other organisms. Bacterially expressed recombinant proteins (rCsGSTo1 and 2) exhibited dehydroascorbate reductase (DHAR) and thioltransferase activities. DHAR activity was higher than thioltransferase activity. They showed weak canonical GST activity toward 1-chloro-2,4-dinitrobenzene. S-hexylglutathione potently and competitively inhibited the active-site at nanomolar concentrations (0.63 and 0.58 nM for rCsGSTo1 and 2). Interestingly, rCsGSTos exhibited high enzyme activity toward mu- and theta-class GST specific substrate, 4-nitrobenzyl chloride. Expression of CsGSTo transcripts and proteins increased beginning in 2-week-old juveniles and reached their highest levels in 4-week-old adults. The proteins were mainly expressed in the elements of the reproductive system, such as vitelline follicles, testes, seminal receptacle, sperm and eggs. Oxidative stressors induced upregulated expression of CsGSTos in these organs. Regardless of oxidative stresses, CsGSTos continued to be highly expressed in eggs. CsGSTo1 or 2 overexpressing bacteria demonstrated high resistance under oxidative killing. CONCLUSIONS: CsGSTos might be critically involved in protection of the reproductive system during maturation of C. sinensis worms and in response to oxidative conditions, thereby contributing to maintenance of parasite fecundity.

Bortezomib attenuates palmitic acid-induced ER stress, inflammation and insulin resistance in myotubes via AMPK dependent mechanism.

Bortezomib is an anti-cancer agent that induces ER stress by inhibiting proteasomal degradation. However, the effects of bortezomib appear to be dependent on its concentration and cellular context. Since ER stress is closely related to type 2 diabetes, the authors examined the effects of bortezomib on palmitic acid (PA)-induced ER stress in C2C12 murine myotubes. At low concentrations (<20nM), bortezomib protected myotubes from PA (750µM)-induced ER stress and inflammation. Either tunicamycin or thapsigargin-induced ER stress was also reduced by bortezomib. In addition, reduced glucose uptake and Akt phosphorylation induced by PA were prevented by co-treating bortezomib (10nM) both in the presence or absence of insulin. These protective effects of bortezomib were found to be associated with reduced JNK phosphorylation. Furthermore, bortezomib-induced AMPK phosphorylation, and the protective effects of bortezomib were diminished by AMPK knockdown, suggesting that AMPK activation underlies the effects of bortezomib. The in vivo administration of bortezomib at nontoxic levels (at 50 or 200µg/kg, i.p.) twice weekly for 5weeks to ob/ob mice improved insulin resistance, increased AMPK phosphorylation, reduced ER stress marker levels, and JNK inhibition in skeletal muscle. The study shows that bortezomib reduces ER stress, inflammation, and insulin resistance in vitro and in vivo, and suggests that bortezomib has novel applications for the treatment of metabolic disorders.

Functionally Expression of Metalloproteinase in Taenia solium Metacestode and Its Evaluation for Serodiagnosis of Cysticercosis.

BACKGROUND: Parasite proteases have important roles in cleavage of host proteins during the invasion of host tissues and participate in the parasite's evasion from the host's immune response. The aim of the present study was to estimate a metalloproteinase properties of Taenia solium metacestode (TsMP) during host-parasite interactions, and evaluate its potential as a serodiagnostic antigen for cysticercosis. METHODS: The cDNA coding for the mature catalytic domain of TsMP was cloned into pGEX-6P-1 expression vector. A recombinant glutathione S-transferase and TsMP fusion protein was induced. After refolding and purification, enzymatic properties of the recombinant metalloproteinase were observed. Immunoblot assay was processed to evaluate its potential as a serodiagnostic antigen for cysticercosis. RESULTS: The recombinant TsMP protein showed proteolytic activity, which preferred host extracellular matrix proteins such as collagen and fibronectin as degradable substrates. In immunoblot assay, 87.5% of sera from patients with cysticercosis showed strong reactivity. In sera from patients with other parasitic infections and from normal controls, it showed high specificity. CONCLUSIONS: TsMP might be involved in the processing of numerous host proteins and play an important role in the parasite life cycle. A single recombinant TsMP antigen could have a potential value for serodiagnosis of cysticercosis.

Phylogenetic characterization of Clonorchis sinensis proteins homologous to the sigma-class glutathione transferase and their differential expression profiles.

Glutathione transferase (GST) is one of the major antioxidant proteins with diverse supplemental activities including peroxidase, isomerase, and thiol transferase. GSTs are classified into multiple classes on the basis of their primary structures and substrate/inhibitor specificity. However, the evolutionary routes and physiological environments specific to each of the closely related bioactive enzymes remain elusive. The sigma-like GSTs exhibit amino acid conservation patterns similar to the prostaglandin D synthases (PGDSs). In this study, we analyzed the phylogenetic position of the GSTs of the biocarcinogenic liver fluke, Clonorchis sinensis. We also observed induction profile of the GSTs in association with the parasite's maturation and in response to exogenous oxidative stresses, with special attention to sigma-class GSTs and PGDSs. The C. sinensis genome encoded 12 GST protein species, which were separately assigned to cytosolic (two omega-, one zeta-, two mu-, and five sigma-class), mitochondrial (one kappa-class), and microsomal (one membrane-associated proteins in eicosanoid and glutathione metabolism-like protein) GST families. Multiple sigma GST (or PGDS) orthologs were also detected in Opisthorchis viverrini. Other trematode species possessed only a single sigma-like GST gene. A phylogenetic analysis demonstrated that one of the sigma GST lineages duplicated in the common ancestor of trematodes were specifically expanded in the opisthorchiids, but deleted in other trematodes. The induction profiles of these sigma GST genes along with the development and aging of C. sinensis, and against various exogenous chemical stimuli strongly suggest that the paralogous sigma GST genes might be undergone specialized evolution to cope with the diverse hostile biochemical environments within the mammalian hepatobiliary ductal system.

An Echinococcus multilocularis Antigen B3 Proteoform That Shows Specific Antibody Responses to Active-Stage Alveolar Echinococcosis.

Alveolar echinococcosis (AE), caused by the Echinococcus multilocularis metacestode, represents one of the most frequently fatal zoonoses. Early diagnosis significantly reduces morbidity and mortality associated with AE. Diagnosis of AE largely depends on a combination of imaging and serological tests due to its minimal clinical manifestations. Several antigens derived from the whole worm and protoscolex have been targeted for AE serodiagnosis, while the antigenic properties of E. multilocularis hydatid fluid (EmHF) are unclear. We observed two AE-specific 6- and 8-kDa antigen proteoforms through an immunoproteome array of the EmHF. We identified these proteins as representing an E. multilocularis antigen B3 (EmAgB3) isoform, and the proteins were shown to be encoded by the same gene. We cloned the gene and expressed the recombinant EmAgB3 protein (rEmAgB3) in Escherichia coli. rEmAgB3 exhibited sensitivity of 90.9% (80/88 cases) and specificity of 98.5% (597/606 samples) by immunoblotting. The positive and negative predictive values were 89.9% and 98.6%, respectively. The protein did not show antibody responses to 33 AE sera collected during posttreatment follow-up monitoring. Mouse sera experimentally infected with AE protoscoleces began to demonstrate specific antibody responses to native and recombinant EmAgB3 6 months after infection. At that stage, fully mature metacestode vesicles that harbored the brood capsule, primary cell, and protoscolex were observed within an AE mass(es). The response declined along with worm degeneration. Our results demonstrate that the immune responses to this EmAgB3 isoform were highly correlated with worm viability accompanied with AE progression. rEmAgB3 is a promising biomarker for serological assessment of AE patients.