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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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Clonorchis sinensis is a carcinogenic human liver fluke, prolonged infection which provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis, and even cholangiocarcinoma (CCA). These effects are driven by direct physical damage caused by the worms, as well as chemical irritation from their excretory-secretory products (ESPs) in the bile duct and surrounding liver tissues. We investigated the C. sinensis ESP-mediated malignant features of CCA cells (HuCCT1) in a three-dimensional microfluidic culture model that mimics an in vitro tumor microenvironment. This system consisted of a type I collagen extracellular matrix, applied ESPs, GFP-labeled HuCCT1 cells and quiescent biliary ductal plates formed by normal cholangiocytes (H69 cells). HuCCT1 cells were attracted by a gradient of ESPs in a concentration-dependent manner and migrated in the direction of the ESPs. Meanwhile, single cell invasion by HuCCT1 cells increased independently of the direction of the ESP gradient. ESP treatment resulted in elevated secretion of interleukin-6 (IL-6) and transforming growth factor-beta1 (TGF-β1) by H69 cells and a cadherin switch (decrease in E-cadherin/increase in N-cadherin expression) in HuCCT1 cells, indicating an increase in epithelial-mesenchymal transition-like changes by HuCCT1 cells. Our findings suggest that C. sinensis ESPs promote the progression of CCA in a tumor microenvironment via the interaction between normal cholangiocytes and CCA cells. These observations broaden our understanding of the progression of CCA caused by liver fluke infection and suggest a new approach for the development of chemotherapeutic for this infectious cancer. [ABSTRACT FROM AUTHOR]

The rapidly evolving cloning and sequencing technologies have enabled understanding of genomic structure of parasite genomes, opening up new ways of combatting parasite-related diseases. To make the most of the exponentially accumulating genomic data, however, it is crucial to analyze the proteins encoded by these genomic sequences. In this study, we adopted an engineered cell-free protein synthesis system for large-scale expression screening of an expression sequence tag (EST) library of Clonorchis sinensis to identify potential antigens that can be used for diagnosis and treatment of clonorchiasis. To allow high-throughput expression and identification of individual genes comprising the library, a cell-free synthesis reaction was designed such that both the template DNA and the expressed proteins were co-immobilized on the same microbeads, leading to microbead-based linkage of the genotype and phenotype. This reaction configuration allowed streamlined expression, recovery, and analysis of proteins. This approach enabled us to identify 21 antigenic proteins. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:832-837, 2017 [ABSTRACT FROM AUTHOR]

Clonorchis sinensis is the causative agent of the life-threatening disease endemic to China, Korea, and Vietnam. It is estimated that about 15 million people are infected with this fluke. C. sinensis provokes inflammation, epithelial hyperplasia, and periductal fibrosis in bile ducts, and may cause cholangiocarcinoma in chronically infected individuals. Accumulation of a large amount of biological information about the adult stage of this liver fluke in recent years has advanced our understanding of the pathological interplay between this parasite and its hosts. However, no developmental gene expression profiles of C. sinensis have been published. In this study, we generated gene expression profiles of three developmental stages of C. sinensis by analyzing expressed sequence tags (ESTs). Complementary DNA libraries were constructed from the adult, metacercaria, and egg developmental stages of C. sinensis. A total of 52,745 ESTs were generated and assembled into 12,830 C. sinensis assembled EST sequences, and then these assemblies were further categorized into groups according to biological functions and developmental stages. Most of the genes that were differentially expressed in the different stages were consistent with the biological and physical features of the particular developmental stage; high energy metabolism, motility and reproduction genes were differentially expressed in adults, minimal metabolism and final host adaptation genes were differentially expressed in metacercariae, and embryonic genes were differentially expressed in eggs. The higher expression of glucose transporters, proteases, and antioxidant enzymes in the adults accounts for active uptake of nutrients and defense against host immune attacks. The types of ion channels present in C. sinensis are consistent with its parasitic nature and phylogenetic placement in the tree of life. We anticipate that the transcriptomic information on essential regulators of development, bile chemotaxis, and physico-metabolic pathways in C. sinensis that presented in this study will guide further studies to identify novel drug targets and diagnostic antigens. Author Summary: Clonorchis sinensis is a significant pathogen that causes clonorchiasis, which is endemic to East Asian countries. This fluke provokes acute inflammation and chronic hyperplasic changes in the biliary tracts. C. sinensis promotes cholangiocarcinoma, and has been classified as a Group 1 biological carcinogen, alongside Opisthorchis viverrini, by the World Health Organization. Recently, transcriptomes for adult liver flukes have been reported with the molecular functionalities predicted on the bases of their transcriptomic data sets. We generated the developmental C. sinensis transcriptome for three different developmental stages, revealing that most functional genes were differentially expressed in each developmental stage; only a small proportion of the expressed genes were shared between the three stages. The developmental transcriptome describes the gene expression landscapes of C. sinensis adults, metacercariae, and eggs, and provides insight into how this fluke adapts to the distinctly different environments provided by its various hosts. We anticipate that the transcriptome will contribute significantly to the identification of intervention points along the developmental stages and allow the exploitation of novel potential targets for diagnostic, drug, and vaccine development purposes. [ABSTRACT FROM AUTHOR]

Abstract: A gene encoding a serine protease inhibitor of Clonorchis sinensis (CsSERPIN) was identified and characterized. CsSERPIN contained an open reading frame of 1158bp that encoded 385 amino acid residues. Sequence analysis of the primary structure of CsSERPIN revealed that it had essential structural motifs including a reactive central loop (RCL), which well conserved in the serine protease inhibitor (serpin) superfamily. CsSERPIN was classified as a member of the ovalbumin-type serpin family on the basis of phylogenetic analysis and the absence of a classical N-terminal signal peptide. Recombinant CsSERPIN showed an inhibitory effect on chymotrypsin in a dose-dependent manner, but did not effectively inhibit trypsin, thrombin, elastases or cathepsin G. Optimal pH values of CsSERPIN were between 7.0 and 9.0, as evidenced by the rapid loss of inhibitory activity under acidic conditions. CsSERPIN was expressed at various developmental stages of the parasite, from eggs to adult worms, but its expression level was higher in eggs and adult worms than in metacercariae and juvenile worms. CsSERPIN was identified in the soluble extract of the parasite, but not in the excretory and secretory products (ESP) or insoluble extract of the parasite. Immunolocalization analysis of CsSERPIN showed that it mainly localized to the eggs and vitelline glands of the adult worm. These results suggest that intracellular CsSERPIN may be possibly involved in maintaining the physiology of eggs as well as in egg production of C. sinensis by regulating endogenous serine proteases. [ABSTRACT FROM AUTHOR]