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Background: Clonorchis sinensis causes chronic cumulative infections in the human hepatobiliary tract and is intimately associated with cholangiocarcinoma. Approximately 35 million people are infected and 600 million people are at risk of infections worldwide. C. sinensis excretory-secretory products (ESP) constitute the first-line effector system affecting the host-parasite interrelationship by interacting with bile fluids and ductal epithelium. However, the secretory behavior of C. sinensis in an environment close to natural host conditions is unclear. C. sinensis differs from Fasciola hepatica in migration to, and maturation in, the hepatic bile duct, implying that protein profile of the ESP of these two trematodes might be different from each other. Methodology/Principal Findings: We conducted systemic approaches to analyze the C. sinensis ESP proteome and the biological reactivity of C. sinensis glutathione transferases (GSTs), such as global expression patterns and induction profiles under oxidative stress and host bile. When we observed ex host excretion behavior of C. sinensis in the presence of 10% host bile, the global proteome pattern was not significantly altered, but the amount of secretory proteins was increased by approximately 3.5-fold. Bioactive molecules secreted by C. sinensis revealed universal/unique features in relation to its intraluminal hydrophobic residing niche. A total of 38 protein spots identified abundantly included enzymes involved in glucose metabolism (11 spots, 28.9%) and diverse-classes of glutathione transferases (GSTs; 10 spots, 26.3%). Cathepsin L/F (four spots, 10.5%) and transporter molecules (three spots, 7.9%) were also recognized. The universal secretory proteins found in other parasites, such as several enzymes involved in glucose metabolism and oxygen transporters, were commonly detected. C. sinensis secreted less cysteine proteases and fatty acid binding proteins compared to other tissue-invading or intravascular trematodes. Interestingly, secretion of a 28 kDa σ-class GST (Cs28σGST3) was significantly affected by the host bile, involving reduced secretion of the 28 kDa species and augmented secretion of Cs28σGST3-related high-molecular-weight 85 kDa protein. Oxidative stressors induced upregulated secretion of 28 kDa Cs28σGST3, but not an 85 kDa species. A secretory 26 kDa μ-class GST (Cs26μGST2) was increased upon treatment with oxidative stressors and bile juice, while another 28 kDa σ-class GST (Cs28σGST1) showed negligible responses. Conclusions/Significance: Our results represent the first analysis of the genuine nature of the C. sinensis ESP proteome in the presence of host bile mimicking the natural host environments. The behavioral patterns of migration and maturation of C. sinensis in the bile ducts might contribute to the secretion of copious amounts of diverse GSTs, but a smaller quantity and fewer kinds of cysteine proteases. The Cs28σGST1 and its paralog(s) detoxify endogenous oxidative molecules, while Cs28σGST3 and Cs26μGST2 conjugate xenobiotics/hydrophobic substances in the extracellular environments, which imply that diverse C. sinensis GSTs might have evolved for each of the multiple specialized functions. [ABSTRACT FROM AUTHOR]

Although fishborne zoonotic trematodes that infect the liver are well documented in Vietnam, intestinal fishborne zoonotic trematodes are unreported. Recent discoveries of the metacercarial stage of these flukes in wild and farmed fish prompted an assessment of their risk to a community that eats raw fish. A fecal survey of 615 persons showed a trematode egg prevalence of 64.9%. Infected persons were treated to expel liver and intestinal parasites for specific identification. The liver trematode Clonorchis sinensis was recovered from 51.5%, but > or =1 of 4 intestinal species of the family Heterophyidae was recovered from 100%. The most numerous were Haplorchis spp. (90.4% of all worms recovered). These results demonstrate that fishborne intestinal parasites are an unrecognized food safety risk in a country whose people have a strong tradition of eating raw fish. [ABSTRACT FROM AUTHOR]

Background: Plasmodium falciparum circumsporozoite protein (PfCSP) is one of the most extensively studied malaria vaccine candidates, but the genetic polymorphism of PfCSP within and among the global P. falciparum population raises concerns regarding the efficacy of a PfCSP-based vaccine efficacy. In this study, genetic diversity and natural selection of PfCSP in Myanmar as well as global P. falciparum were comprehensively analysed. Methods: Blood samples were collected from 51 P. falciparum infected Myanmar patients. Fifty-one full-length PfCSP genes were amplified from the blood samples through a nested polymerase chain reaction, cloned into a TA cloning vector, and then sequenced. Polymorphic characteristics and natural selection of Myanmar PfCSP were analysed using the DNASTAR, MEGA6, and DnaSP programs. Polymorphic diversity and natural selection in publicly available global PfCSP were also analysed. Results: The N-terminal and C-terminal non-repeat regions of Myanmar PfCSP showed limited genetic variations. A comparative analysis of the two regions in global PfCSP displayed similar patterns of low genetic diversity in global population, but substantial geographic differentiation was also observed. The most notable polymorphisms identified in the N-terminal region of global PfCSP were A98G and 19-amino acid length insertion in global population with different frequencies. Major polymorphic characters in the C-terminal region of Myanmar and global PfCSP were found in the Th2R and Th3R regions, where natural selection and recombination occurred. The central repeat region of Myanmar PfCSP was highly polymorphic, with differing numbers of repetitive repeat sequences NANP and NVDP. The numbers of the NANP repeats varied among global PfCSP, with the highest number of repeats seen in Asian and Oceanian PfCSP. Haplotype network analysis of global PfCSP revealed that global PfCSP clustered into 103 different haplotypes with geographically-separated populations. Conclusion: Myanmar and global PfCSP displayed genetic diversity. N-terminal and C-terminal non-repeat regions were relatively conserved, but the central repeat region displayed high levels of genetic polymorphism in Myanmar and global PfCSP. The observed geographic pattern of genetic differentiation and the points of evidence for natural selection and recombination suggest that the functional consequences of the polymorphism should be considered for developing a vaccine based on PfCSP. [ABSTRACT FROM AUTHOR]

Background: The C-terminal 42 kDa region of merozoite surface protein-1 of Plasmodium falciparum (PfMSP-142) is the target of an immune response. It has been recognised as one of the promising candidate antigens for a blood-stage malaria vaccine. Genetic structure of PfMSP-142 has been considered to be largely conserved in the P. falciparum population. However, only limited information is currently available. This study aimed to analyse genetic diversity and the effect of natural selection on PfMSP-142 among the Myanmar P. falciparum population and compare them with publicly available PfMSP-142 from global P. falciparum populations. Methods: A total of 69 P. falciparum clinical isolates collected from Myanmar malaria patients in Upper Myanmar in 2015 were used. The PfMSP-142 region was amplified by polymerase chain reaction, cloned and sequenced. Genetic structure and natural selection of this region were analysed using MEGA4 and DnaSP programs. Polymorphic nature and natural selection in global PfMSP-142 were also investigated. Results: All three allele types (MAD20, K1, and RO33) of PfMSP-142 were identified in Myanmar isolates of P. falciparum. Myanmar PfMSP-142 displayed genetic diversity. Most polymorphisms were scattered in blocks 16 and 17. Polymorphisms observed in Myanmar PfMSP-142 showed a similar pattern to those of global PfMSP-142; however, they were not identical to each other. Genetic diversity of Myanmar PfMSP-142 was relatively lower than that of PfMSP-142 from different geographical regions. Evidence of natural selection and recombination were found. Comparative analysis of genetic polymorphism and natural selection in the global PfMSP-142 population suggested that this region was not tightly conserved in global PfMSP-142 as previously thought and is under the complicated influence of natural selection and recombination. Conclusions: Global PfMSP-142 revealed limited, but non-negligible, genetic diversity by allele types and geographical origins. Complicated natural selection and potential recombination might have occurred in global PfMSP-142. Comprehensive monitoring of genetic diversity for global PfMSP-142 would be needed to better understand the polymorphic nature and evolutionary aspect of PfMSP-142 in the global P. falciparum population. More thought would be necessary for designing a vaccine based on PfMSP-142. [ABSTRACT FROM AUTHOR]

We performed an epidemiologic survey of opisthorchiasis in Yangon, Myanmar. The fecal egg-positive rate of residents was 0.7%, and we recovered an adult fluke after chemotherapy and purging of an egg-positive resident. We detected Opisthorchis viverrini metacercariae in freshwater fish. We found the Yangon area to have low-grade endemicity of opisthorchiasis. [ABSTRACT FROM AUTHOR]

Clonorchis sinensis is a carcinogenic human liver fluke. Its infection promotes persistent oxidative stress and chronic inflammation environments in the bile duct and surrounding liver tissues owing to direct contact with worms and their excretory–secretory products (ESPs), provoking epithelial hyperplasia, periductal fibrosis, and cholangiocarcinogenesis. We examined the reciprocal regulation of two ESP-induced redox-active proteins, NF-κB and peroxiredoxin 6 (Prdx6), during C. sinensis infection. Prdx6 overexpression suppressed intracellular free-radical generation by inhibiting NADPH oxidase2 and inducible nitric oxide synthase activation in the ESP-treated cholangiocarcinoma cells, substantially attenuating NF-κB-mediated inflammation. NF-κB overexpression decreased Prdx6 transcription levels by binding to two κB sites within the promoter. This transcriptional repression was compensated for by other ESP-induced redox-active transcription factors, including erythroid 2-related factor 2 (Nrf2), hypoxia inducible factor 1α (HIF1α), and CCAAT/enhancer-binding protein β (C/EBPβ). Distribution of immunoreactive Prdx6 and NF-κB was distinct in the early stages of infection in mouse livers but shared concomitant localization in the later stages. The intensity and extent of their immunoreactive staining in infected mouse livers are proportional to lesion severity and infection duration. The constitutive elevations of Prdx6 and NF-κB during C. sinensis infection may be associated with more severe persistent hepatobiliary abnormalities mediated by clonorchiasis. [ABSTRACT FROM AUTHOR]

This study is a contribution to the molecular taxonomy and epidemiology of heterophyid (Trematoda: Heterophyidae) metacercariae found in the muscle of Mugilidae (Osteichthyes) from Sardinia (western Mediterranean Sea). Sixty specimens of mugilids (13 Chelon labrosus, 18 Liza aurata, 6 Liza ramada, 8 Liza saliens, 15 Mugil cephalus) were examined and 17,899 metacercariae isolated in 95 % of the hosts. Four types of metacercariae were identified: Heterophyes sp. ( n = 14,113), Heterophyes sp. -small (1225), Stictodora sp. (1606), and Ascocotyle ( Phagicola) sp. (955). The experimental infection of a hamster with Heterophyes sp. metacercariae produced six adults identified as Heterophyes heterophyes and two as Heterophyes cf. nocens. The morphology of Heterophyes sp. -small metacercariae matched with that of Heterophyes dispar. The sequence analysis of the ITS2 and 28S portions of rDNA confirmed the morphological identification of metacercariae, showing four clusters. All adults grouped together with the Heterophyes sp. metacercariae, whereas adults of Heterophyes nocens from Korea clustered separately, showing that this species is distinguished from H. heterophyes, and suggesting caution in the exclusive use of the number of rodlets of the genital sucker to separate the two species. The presence of metacercariae was high in all hosts; the highest prevalence is of Heterophyes sp. (prevalence ≥78 %; mean intensity ≥135 metacercariae/100 g muscle), and the most heavily infected host is M. cephalus (prevalence = 100 %; mean intensity = 841 metacercariae/100 g muscle). [ABSTRACT FROM AUTHOR]

Clonorchis sinensis is a high-risk pathogenic helminth that strongly provokes inflammation, epithelial hyperplasia, periductal fibrosis, and even cholangiocarcinoma in chronically infected individuals. Chronic inflammation is associated with an increased risk of various cancers due to the disruption of redox homeostasis. Accordingly, the present study was conducted to examine the time course relationship between histopathological changes and the appearance of oxidative stress markers, including lipid peroxidation, enzymes involved in lipid peroxidation, and mutagenic DNA adducts in the livers of mice infected with C. sinensis , as well as proinflammatory cytokines in infected mouse sera. Histopathological phenotypes such as bile duct epithelial hyperplasia, periductal fibrosis, edema and inflammatory infiltration increased in infected livers in a time-dependent manner. Intense immunoreactivity of lipid peroxidation products (4-hydroxy-2-nonenal; malondialdehyde), cyclooxygenase-2, 5-lipoxygenase and 8-oxo-7,8-dihydro-2′-deoxyguanosine were concomitantly observed in these injured regions. We also found elevated expressions of cyclooxygenase-2 and 5-lipoxygenase in C. sinensis excretory-secretory product-treated cholangiocarcinoma cells. Moreover, the levels of proinflammatory cytokines such as TNF-α, ILβ-1 and IL-6 were differentially upregulated in infected sera. With regard to oxidative stress-mediated carcinogenesis, our findings suggest that C. sinensis infestation may disrupt host redox homeostasis, creating a damaging environment that favors the development of advanced hepatobiliary diseases such as clonorchiasis-associated cholangiocarcinoma. [ABSTRACT FROM AUTHOR]

A complementary DNA (cDNA) encoding a glucose transporter of Clonorchis sinensis (CsGLUT) was isolated from the adult C. sinensis cDNA library. The open reading frame of CsGLUT cDNA consists of 1653 base pairs that encode a 550-amino acid residue protein. Hydropathy analysis suggested that CsGLUT possess 12 putative membrane-spanning domains. The Northern blot analysis result using poly(A)RNA showed a strong band at ~2.1 kb for CsGLUT. When expressed in Xenopus oocytes, CsGLUT mediated the transport of radiolabeled deoxy- d-glucose in a time-dependent but sodium-independent manner. Concentration-dependency results showed saturable kinetics and followed the Michaelis-Menten equation. Nonlinear regression analyses yielded a Km value of 588.5 ± 53.0 μM and a Vmax value of 1500.0 ± 67.5 pmol/oocyte/30 min for [1,2-H]2-deoxy- d-glucose. No trans-uptakes of bile acid (taurocholic acid), amino acids (tryptophan and arginine), or p-aminohippuric acid were observed. CsGLUT-mediated transport of deoxyglucose was significantly and concentration-dependently inhibited by radio-unlabeled deoxyglucose and d-glucose. 3- O-Methylglucose at 10 and 100 μM inhibited deoxyglucose uptake by ~50 % without concentration dependence. No inhibitory effects by galactose, mannose, and fructose were observed. This work may contribute to the molecular biological study of carbohydrate metabolism and new drug development of C. sinensis. [ABSTRACT FROM AUTHOR]

Asparaginyl endopeptidases (AEP: EC 3.4.22.34) are a family of cysteine proteases classified into the MEROPS clan CD, family C13. In this study, we characterized the biochemical and antigenic properties of an AEP of Clonorchis sinensis (CsAEP). The recombinant CsAEP showed hydrolytic activity at pH values ranging from acidic to neutral with optimum activity at pH 6.0. While the recombinant CsAEP was stable at neutral pHs, it was unstable at acidic pHs and resulted in loss of enzymatic activity. The recombinant enzyme was effectively inhibited by iodoacetic acid and N -ethylmaleimide, but not by E-64. The partially purified native CsAEP showed biochemical properties similar to the recombinant enzyme. Native CsAEP is likely to be cleaved into an N-terminal mature enzyme and a C-terminal fragment via autocatalytic activation at acidic pHs. Polyclonal antibody raised against the recombinant CsAEP recognized three forms of CsAEP, proenzyme, the N-terminal mature enzyme and the C-terminal fragment, in the worm extract (WE) of C. sinensis . However, only the C-terminal fragment was mainly found in the excretory and secretory (ES) products of the parasite. Strong CsAEP activity was found in the WE, but only a trace level of CsAEP activity was detected in the ES products of the parasite. CsAEP was expressed in various developmental stages of C. sinensis , from metacercariae to adults, and was found to be localized in the intestine of the parasite as well as in intestinal contents. Sera from rats experimentally infected with C. sinensis reacted with CsAEP beginning 4 weeks after infection. These results suggest that CsAEP is a gut-associated enzyme synthesized in the intestine of C. sinensis and subsequently secreted into the intestinal lumen of the parasite. [ABSTRACT FROM AUTHOR]

Paramyosin of Clonorchis sinensis (CsPmy) is a myofibrillar protein localized in subtegumental muscle, tegument, and the muscle layer surrounding the intestine of the parasite. Previously, we have identified that CsPmy reacted with sera of human clonorchiasis and this protein had a potential as a candidate antigen for serodiagnosis of clonorchiasis. However, we also found that CsPmy is able to bind to human immunoglobulin G (IgG) in non-specific manners, which can affect the diagnostic value of the protein. Here, we mapped CsPmy-specific IgG binding site on CsPmy to analyze the putative epitopes recognized by CsPmy-specific IgG in sera of human clonorchiasis. The fragmental expression of CsPmy followed by immunoblot analyses with sera from patients with clonorchiasis and non-specific human IgG revealed that the middle portion of CsPmy (CsPmyC: 301–600 amino acid residues) had epitopes responsible for CsPmy-specific IgG recognition. The precise CsPmy-specific IgG binding site was further narrowed down to a fragment (CsPmyC-2), which harbors 151 amino acid residues (375–525) of CsPmy. Specific antibodies for CsPmyC-2 were produced in rats after two-weeks of post-experimental infection. The CsPmyC-2 showed low levels of cross reactivity against the sera from patients with other helminth parasites. Our results suggested that CsPmyC-2 has real epitopes recognized by CsPmy-specific IgG in sera of human clonorchiasis and the fragment can be useful as a reliable serodiagnostic antigen to develop a serodiagnostic method for clonorchiasis. [ABSTRACT FROM AUTHOR]

Methionine aminopeptidases (MetAPs), ubiquitous enzymes that play an important role in nascent protein maturation, have been recognized as attractive targets for the development of drugs against pathogenic protozoa including Plasmodium spp. Here, we characterized partial biochemical properties of a type I MetAP of Plasmodium vivax (PvMetAP1). PvMetAP1 had the typical amino acid residues essential for metal binding and substrate binding sites, which are well conserved in the type I MetAP family enzymes. Recombinant PvMetAP1 showed activity in a broad range of neutral pHs, with optimum activity at pH 7.5. PvMetAP1 was stable under neutral and alkaline pHs, but was relatively unstable under acidic conditions. PvMetAP1 activity was highly increased in the presence of Mn 2+ , and was effectively inhibited by a metal chelator, EDTA. Fumagillin and aminopeptidase inhibitors, amastatin and bestatin, also showed an inhibitory effect on PvMetAP1. The enzyme had a highly specific hydrolytic activity for N-terminal methionine. These results collectively suggest that PvMetAP1 belongs to the family of type I MetAPs and may play a pivotal role for the maintenance of P. vivax physiology by mediating protein maturation and processing of the parasite. [ABSTRACT FROM AUTHOR]

Abstract: Otubains are a recently discovered family of cysteine proteases that participate in the ubiquitin pathway. Here, we partially characterized the biochemical properties of a cysteine protease of Cryptosporidium parvum, which is closely related to otubains. The gene encoding otubain-like cysteine protease of C. parvum (CpOTU) contained the aspartate, cysteine and histidine residues that form the catalytic triad of otubains. The modified ubiquitin-associated domain and LxxL motif were identified in CpOTU. The recombinant CpOTU showed the isopeptidase activity at neutral pH values and its activity was effectively inhibited by ubiquitin aldehyde, N-ethylmaleimide and iodoacetic acid. Interestingly, CpOTU had an unusual C-terminal extension of 217 amino acids compared to mammalian otubains, and the C-terminal extension is essential for the activity of the enzyme. Expression of CpOTU peaked in the oocyst stage of the parasite, which suggested its potential physiological role for the oocyst stage. [Copyright &y& Elsevier]

CsStefin-2, the second cysteine protease inhibitor of Clonorchis sinensis, was identified and characterized. CsStefin-2 is a cysteine protease inhibitor that belongs to family 1 stefins based on its phylogenetic and structural properties. However, CsStefin-2 had a QIVSG cystatin motif distinct from the common QVVAG cystatin motif that is well conserved in family 1 stefins. Mutagenesis analysis revealed that the two amino acid substitutions in the QIVSG cystatin motif of CsStefin-2 did not affect its inhibitory activity. Molecular modeling also indicated that no critical change was induced in the interaction between CsStefin-2 and its target enzyme. CsStefin-2 showed broad inhibitory activities against several cysteine proteases, including human cathepsins B and L, papain, and cathepsin Fs of C. sinensis (CsCFs), and effectively inhibited the autocatalytic maturation of CsCF-6. Native CsStefin-2 was assembled into a homo-tetramer, in which intermolecular disulfide bonds are not involved in the assembly of the tetramer. CsStefin-2 was expressed throughout the various developmental stages of the parasite and was localized in the intestinal epithelium, where CsCFs are actively synthesized. These results suggest that CsStefin-2 is the second active cysteine protease inhibitor of C. sinensis that shares functional redundancy with CsStefin-1 to modulate the activity and processing of CsCFs. [ABSTRACT FROM AUTHOR]

Highlights: [•] Four novel genes encoding Clonorchis sinensis tyrosinases (CsTYR) were characterised. [•] CsTYRs showed high reactivity against diphenol compounds, but not toward monophenol compounds. [•] CsTYR expression coordinated with the sexual maturation of the worm in the vitelline glands and intrauterine immature eggs. [•] In vitro treatment of copper chelator, diethyldithiocarbamic acid, inhibited generation of phenotypically normal egg. [•] The duplication of trematode TYR genes might relate to increased genic dosage or redundancy. [ABSTRACT FROM AUTHOR]

Background: Clonorchis sinensis causes chronic cumulative infections in the human hepatobiliary tract and is intimately associated with cholangiocarcinoma. Approximately 35 million people are infected and 600 million people are at risk of infections worldwide. C. sinensis excretory-secretory products (ESP) constitute the first-line effector system affecting the host-parasite interrelationship by interacting with bile fluids and ductal epithelium. However, the secretory behavior of C. sinensis in an environment close to natural host conditions is unclear. C. sinensis differs from Fasciola hepatica in migration to, and maturation in, the hepatic bile duct, implying that protein profile of the ESP of these two trematodes might be different from each other. Methodology/Principal Findings: We conducted systemic approaches to analyze the C. sinensis ESP proteome and the biological reactivity of C. sinensis glutathione transferases (GSTs), such as global expression patterns and induction profiles under oxidative stress and host bile. When we observed ex host excretion behavior of C. sinensis in the presence of 10% host bile, the global proteome pattern was not significantly altered, but the amount of secretory proteins was increased by approximately 3.5-fold. Bioactive molecules secreted by C. sinensis revealed universal/unique features in relation to its intraluminal hydrophobic residing niche. A total of 38 protein spots identified abundantly included enzymes involved in glucose metabolism (11 spots, 28.9%) and diverse-classes of glutathione transferases (GSTs; 10 spots, 26.3%). Cathepsin L/F (four spots, 10.5%) and transporter molecules (three spots, 7.9%) were also recognized. The universal secretory proteins found in other parasites, such as several enzymes involved in glucose metabolism and oxygen transporters, were commonly detected. C. sinensis secreted less cysteine proteases and fatty acid binding proteins compared to other tissue-invading or intravascular trematodes. Interestingly, secretion of a 28 kDa σ-class GST (Cs28σGST3) was significantly affected by the host bile, involving reduced secretion of the 28 kDa species and augmented secretion of Cs28σGST3-related high-molecular-weight 85 kDa protein. Oxidative stressors induced upregulated secretion of 28 kDa Cs28σGST3, but not an 85 kDa species. A secretory 26 kDa μ-class GST (Cs26μGST2) was increased upon treatment with oxidative stressors and bile juice, while another 28 kDa σ-class GST (Cs28σGST1) showed negligible responses. Conclusions/Significance: Our results represent the first analysis of the genuine nature of the C. sinensis ESP proteome in the presence of host bile mimicking the natural host environments. The behavioral patterns of migration and maturation of C. sinensis in the bile ducts might contribute to the secretion of copious amounts of diverse GSTs, but a smaller quantity and fewer kinds of cysteine proteases. The Cs28σGST1 and its paralog(s) detoxify endogenous oxidative molecules, while Cs28σGST3 and Cs26μGST2 conjugate xenobiotics/hydrophobic substances in the extracellular environments, which imply that diverse C. sinensis GSTs might have evolved for each of the multiple specialized functions. Author Summary: Clonorchis sinensis is a trematode parasite that infects the hepatobiliary ducts of the mammals including humans. Approximately 35 million people are infected and 600 million people are at risk of infections. Epidemiological studies have convincingly demonstrated relationships between clonorchiasis and cholangiocarcinoma. C. sinensis is a Group 1 biological carcinogen. C. sinensis excretory-secretory products (ESP) constitute the first-line effector system affecting the host-parasite interrelationship. We observed global expression pattern of C. sinensis ESP in the presence of host bile/oxidative stresses, which mimics natural host environments. The secretory proteome displayed common and unique features, which might be related to its habitat in the definitive host. The universal proteins were found to be several enzymes involved in glucose metabolism, glutathione transferases (GSTs) and oxygen transporters. C. sinensis differentially regulated the secretion of diverse GSTs in response to bile and oxidative stressors, which suggested that these enzymes are importantly involved in the protection from immune cell-derived oxidizing molecules and detoxification of hydrophobic substances. C. sinensis secreted less cysteine proteases, which play roles in tissue migration and immune evasion, compared to other tissue-invasive or intravascular trematodes. Our data suggest strongly that different GSTs might have differentially evolved with their specialized functions to cope with stressful conditions and ensure parasite's long-standing survival in the hosts. [ABSTRACT FROM AUTHOR]

Abstract: Plasmodium vivax Duffy binding protein (PvDBP) is a micronemal type I membrane protein that plays an essential role in erythrocyte invasion of merozoites. PvDBP is a prime blood stage vaccine candidate antigen against P. vivax, but its polymorphic nature represents a major obstacle to the successful design of a protective vaccine against vivax malaria. In this study, we analyzed the genetic polymorphism and natural selection at the N-terminal cysteine-rich region of PvDBP (PvDBPII) among 70 P. vivax isolates collected from Korean patients during 2005–2010. Seventeen single nucleotide polymorphisms (SNP), which resulted in 14 non-synonymous and 3 synonymous mutations, were found in PvDBPII among the Korean P. vivax isolates. Sequence analyses revealed that 13 different PvDBPII haplotypes, which were clustered into 3 distinct clades, were identified in Korean P. vivax isolates. The difference between the rates of nonsynomyous and synonymous mutations suggested that the region has evolved under natural selection. High selective pressure preferentially acted on regions identified or predicted to be B- and T-cell epitopes and MHC binding regions of PvDBPII. Recombination may also contribute to genetic diversity of PvDBPII. Our results suggest that PvDBPII of Korean P. vivax isolates display a limited genetic polymorphism and are under selective pressure. These results have significant implications for understanding the nature of the P. vivax population circulating in Korea and provide useful information for development of malaria vaccines based on this antigen. [Copyright &y& Elsevier]

Apical membrane antigen-1 of Plasmodium falciparum (PfAMA-1) is a leading malaria vaccine candidate antigen. However, the genetic diversity of pfama-1 and associated antigenic variation in global P. falciparum field isolates are major hurdles to the design of an efficacious vaccine formulated with this antigen. Here, we analyzed the genetic structure and the natural selection of pfama-1 in the P. falciparum population of Vietnam. A total of 37 distinct haplotypes were found in 131 P. falciparum Vietnamese isolates. Most amino acid changes detected in Vietnamese pfama-1 were localized in the ectodomain, domains I, II, and III. Overall patterns of major amino acid changes in Vietnamese pfama-1 were similar to those of global pfama-1, but the frequencies of the amino acid changes slightly differed by country. Novel amino acid changes were also identified in Vietnamese pfama-1. Vietnamese pfama-1 revealed relatively lower genetic diversity than currently analyzed pfama-1 in other geographical regions, and suggested a distinct genetic differentiation pattern. Evidence for natural selection was detected in Vietnamese pfama-1, but it showed purifying selection unlike the global pfama-1 analyzed so far. Recombination events were also found in Vietnamese pfama-1. Major amino acid changes that were commonly identified in global pfama-1 were mainly localized to predicted B-cell epitopes, RBC-binding sites, and IUR regions. These results provide important information for understanding the genetic nature of the Vietnamese pfama-1 population, and have significant implications for the design of a vaccine based on PfAMA-1. [ABSTRACT FROM AUTHOR]

Abstract: Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a major antioxidant enzyme and may protect against lipid hydroperoxidation in biomembranes. We isolated full-length cDNA sequences encoding four different PHGPxs from a causative agent of cholangiocarcinoma, Clonorchis sinensis (CsGPx1, CsGPx2, CsGPx3 and CsGPx4). These sequences contained an in-frame TGA codon for selenocysteine (Sec) and a concurrent Sec insertion sequence in their 3′-untranslated regions. The open reading frames were composed of six exons in the chromosomal segments of CsGPx1 (7705bp), CsGPx2 (5871bp) and CsGPx3 (3867bp) and five exons in CsGPx4 (5655bp). The positions of these introns were tightly conserved between the trematode and vertebrate PHGPx genes. Oxidative stimulation of viable worms with H2O2 or paraquat resulted in 1.5- to 2-fold induction of the GPx activity. The CsGPx proteins were specifically localised in vitellocytes within vitelline follicles and premature eggs in the proximal uterus. In the eggs, glutathione, an electron donor for GPx, was co-localised with the CsGPx proteins, while thioredoxin, which is preferred by peroxiredoxin, was principally detected in the extracellular space between the embryonic cell mass and an eggshell. Our data may suggest a concerted or a specialised function between a thioredoxin-dependent enzyme(s) and GPx in protecting against H2O2-derived damage during maturation of the embryo and formation of the eggshell, in these catalase-lacking trematode parasites. The uniquely conserved genomic organisation and Sec-dependency amongst trematode and vertebrate PHGPx homologues will also provide insight into the evolutionary episode and functional/biochemical diversification of GPx proteins. [Copyright &y& Elsevier]

Background: Circumsporozoite surface protein (CSP) of malaria parasites has been recognized as one of the leading vaccine candidates. Clinical trials of vaccines for vivax malaria incorporating Plasmodium vivax CSP (PvCSP) have demonstrated their effectiveness in preventing malaria, at least in part. However, genetic diversity of pvcsp in the natural population remains a major concern. Methods: A total of 171 blood samples collected from patients infected with Plasmodium vivax in Myanmar were analysed in this study. The pvcsp was amplified by polymerase chain reaction, followed by cloning and sequencing. Polymorphic characteristics and natural selection of pvcsp population in Myanmar were analysed using DNASTAR, MEGA6 and DnaSP programs. The polymorphic pattern and natural selection of publicly accessible global pvcsp sequences were also comparatively analysed. Results: Myanmar pvcsp sequences were divided into two subtypes VK210 and VK247 comprising 143 and 28 sequences, respectively. The VK210 subtypes showed higher levels of genetic diversity and polymorphism than the VK247 subtypes. The N-terminal non-repeat region of pvcsp displayed limited genetic variations in the global population. Different patterns of octapeptide insertion (ANKKAEDA in VK210 and ANKKAGDA in VK247) and tetrapeptide repeat motif (GGNA) were identified in the C-terminal region of global pvcsp population. Meanwhile, the central repeat region (CRR) of Myanmar and global pvcsp, both in VK210 and VK247 variants, was highly polymorphic. The high level of genetic diversity in the CRR has been attributed to the different numbers, types and combinations of peptide repeat motifs (PRMs). Interestingly, 27 and 5 novel PRMs were found in Myanmar VK210 and VK247 variants, respectively. Conclusion: Comparative analysis of the global pvcsp population suggests a complex genetic profile of pvcsp in the global population. These results widen understanding of the genetic make-up of pvcsp in the global P. vivax population and provide valuable information for the development of a vaccine based on PvCSP. [ABSTRACT FROM AUTHOR]