BACKGROUND: Serpins are a superfamily of serine peptidase inhibitors that participate in the regulation of many physiological and cell peptidase-mediated processes in all organisms (e.g. in blood clotting, complement activation, fibrinolysis, inflammation, and programmed cell death). It was postulated that in the blood-feeding members of the monogenean family Diplozoidae, serpins could play an important role in the prevention of thrombus formation, activation of complement, inflammation in the host, and/or in the endogenous regulation of protein degradation. RESULTS: In silico analysis showed that the DNA and primary protein structures of serpin from Eudiplozoon nipponicum (EnSerp1) are similar to other members of the serpin superfamily. The inhibitory potential of EnSerp1 on four physiologically-relevant serine peptidases (trypsin, factor Xa, kallikrein, and plasmin) was demonstrated and its presence in the worm's excretory-secretory products (ESPs) was confirmed. CONCLUSION: EnSerp1 influences the activity of peptidases that play a role in blood coagulation, fibrinolysis, and complement activation. This inhibitory potential, together with the serpin's presence in ESPs, suggests that it is likely involved in host-parasite interactions and could be one of the molecules involved in the control of feeding and prevention of inflammatory responses.

• MeSH
• DNA helmintů chemie   MeSH
• fylogeneze MeSH
• infekce červy třídy Trematoda parazitologie   veterinární   MeSH
• inhibitory serinových proteinas chemie   genetika   izolace a purifikace   metabolismus   MeSH
• kapři parazitologie   MeSH
• nemoci ryb parazitologie   MeSH
• počítačová simulace MeSH
• polymerázová řetězová reakce MeSH
• rekombinantní proteiny genetika   izolace a purifikace   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• serpiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• Trematoda chemie   klasifikace   enzymologie   genetika   MeSH
• žábry parazitologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Eudiplozoon nipponicum (Goto, 1891) Khotenovsky, 1985 (Monogenea: Diplozoidae), is known to parasitise Cyprinus carpio Linnaeus and species of Carassius. In this study, we conducted a taxonomic re-examination of E. nipponicum using genetic analysis and morphological comparisons from different host species from a single water system. rDNA nucleotide sequences of the internal transcription spacer 2 (ITS-2) region (645 bp) showed interspecific-level genetic differences among diplozoids from species of Carassius and C. carpio (p-distance: 3.1-4.0%) but no difference among those from different species of Carassius (0-0.4%) or between those from C. carpio collected in Asia and Europe (0-1.1%). Large variation was observed among 346 bp cytochrome c oxidase subunit I (COI) sequences (0.3-16.0 %); the topology of the phylogenetic tree showed no relationship to host genera or geographical regions of origin. Morphological observation showed that average clamp size of diplozoids from C. carpio was larger than those from Carassius spp. The number of folds on the hindbody was 10-25 for diplozoids from C. carpio and 12-19 for those from Carassius spp. Thus, our ITS-2 sequence and morphological comparison results indicate that diplozoids from C. carpio and species of Carassius belong to different species. The scientific name E. nipponicum should be applied to the species infected to the type host, Carassius sp. of Nakabo (2013) (Japanese name ginbuna). The diplozoid infecting C. carpio (Eurasian type) should be established as a new species: Eudiplozoon kamegaii sp. n. A neotype of E. nipponicum is designated in this report because the original E. nipponicum specimens are thought to have been lost.

• MeSH
• fylogeneze MeSH
• infekce červy třídy Trematoda epidemiologie   parazitologie   veterinární   MeSH
• jezera parazitologie   MeSH
• kapři * MeSH
• nemoci ryb epidemiologie   parazitologie   MeSH
• ribozomální DNA analýza   MeSH
• Trematoda anatomie a histologie   klasifikace   genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Japonsko MeSH

In parasitic flatworms, acid endopeptidases are involved in crucial processes, including digestion, invasion, interactions with the host immune system, etc. In haematophagous monogeneans, however, no solid information has been available about the occurrence of these enzymes. Here we aimed to identify major cysteine and aspartic endopeptidase activities in Eudiplozoon nipponicum, an invasive haematophagous parasite of common carp. Employing biochemical, proteomic and molecular tools, we found that cysteine peptidase activities prevailed in soluble protein extracts and excretory/secretory products (ESP) of E. nipponicum; the major part was cathepsin L-like in nature supplemented with cathepsin B-like activity. Significant activity of the aspartic cathepsin D also occurred in soluble protein extracts. The degradation of haemoglobin in the presence of ESP and worm protein extracts was completely inhibited by a combination of cysteine and aspartic peptidase inhibitors, and diminished by particular cathepsin L, B and D inhibitors. Mass spectrometry revealed several tryptic peptides in ESP matching to two translated sequences of cathepsin L genes, which were amplified from cDNA of E. nipponicum and bioinformatically annotated. The dominance of cysteine peptidases of cathepsin L type in E. nipponicum resembles the situation in, e.g. fasciolid trematodes.

• MeSH
• chromatografie kapalinová MeSH
• cysteinové proteasy metabolismus   MeSH
• endopeptidasy chemie   metabolismus   MeSH
• fluorescenční barviva metabolismus   MeSH
• inhibitory proteas farmakologie   MeSH
• kathepsin B metabolismus   MeSH
• kathepsin D metabolismus   MeSH
• kathepsin L genetika   metabolismus   MeSH
• komplementární DNA chemie   MeSH
• koncentrace vodíkových iontů MeSH
• peptidy metabolismus   MeSH
• ploštěnci enzymologie   genetika   MeSH
• polymerázová řetězová reakce metody   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• sekvenční seřazení MeSH
• tandemová hmotnostní spektrometrie MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Ectoparasitic monogeneans of the family Diplozoidae have direct and monoxenous life cycle. The cytogenetics of monogeneans in general and diplozoids in particular, is a relatively underexplored area. This is why each new detailed description of a karyotype provides significant information about the evolution of monogenean chromosomes and contributes to a better understanding of phylogenetic relationships within this group. This study offers new data on the chromosomes of Eudiplozoon nipponicum, an invasive parasite of the common carp. This species' karyotype consists of seven pairs of telocentric chromosomes (2n = 14 t). After DAPI staining, we marked heterochromatin blocks on all chromosomes in the pericentromeric region. Silver staining (AgNO3) and staining with fluorescent dye YOYO-1 revealed the presence of one large active nucleolus. Fluorescent in situ hybridization with an 18S rDNA probe revealed one cluster of ribosomal genes at the terminal part of the long arms of chromosome pair No. 7. We compared our results with studies on the phylogenetic relationships of diplozoids which applied a combination of molecular methods and classical morphological characterization and found that the results of our cytogenetic analysis are consistent with the hypothesis that E. nipponicum is more basal member of the family Diplozoidae.

• MeSH
• barvení a značení MeSH
• buněčné dělení * MeSH
• cytogenetické vyšetření MeSH
• fylogeneze MeSH
• hybridizace in situ fluorescenční MeSH
• kapři parazitologie   MeSH
• karyotyp MeSH
• RNA ribozomální 18S genetika   MeSH
• spermatocyty fyziologie   MeSH
• Trematoda genetika   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Developmental stages of the diplozoid monogenean Eudiplozoon nipponicum, comprising oncomiracidium, diporpa, juvenile, and adult, were investigated using light and scanning electron microscopy in conjunction with confocal scanning laser microscopy in order to examine body organization and identify explicit morphological adaptations to the ectoparasitic life in each stage. The parasite exhibits a complex digestive tract well equipped for hematophagous feeding. It consists of a mouth opening with prominent buccal suckers, eversible pharynx with adjacent glandular structures, and a blind-ending gut with cecal lining. Glandulo-muscular organs, located apically and opened into the mouth corner, are considered to be a part of the digestive tract. Based on our observations of pharynx eversion and in light of the presence of several glandular or gland-like structures, we propose a new hypothesis on the possibility of extracorporeal digestion of this parasite. The hindbody bears an attachment apparatus, comprising haptor, lobular extensions, and tegumental folds, responsible for the parasite's firm attachment to the host gills. The possibility of buccal suckers assisting in the parasite's translocation while searching for an optimal niche or their temporary attachment function during feeding is discussed. The body of each compound adult (i.e., permanent copula) is almost completely filled by two complete reproductive tracts comprising the female as well as male organs. Such a reproductive strategy, in which two independent heterogenic individuals fuse into a single hermaphrodite organism without the need to search for mating partner, represents a high specialization of diplozoids to their parasitic life.

• MeSH
• fyziologická adaptace MeSH
• hermafroditické organismy fyziologie   MeSH
• infestace ektoparazity parazitologie   patofyziologie   MeSH
• interakce hostitele a parazita fyziologie   MeSH
• kapři parazitologie   MeSH
• mikroskopie elektronová rastrovací MeSH
• parazitární onemocnění kůže parazitologie   patofyziologie   MeSH
• ploštěnci anatomie a histologie   fyziologie   ultrastruktura   MeSH
• rozmnožování fyziologie   MeSH
• stadia vývoje fyziologie   MeSH
• stravovací zvyklosti MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Ectoparasites from the family Diplozoidae (Platyhelminthes, Monogenea) belong to obligate haematophagous helminths of cyprinid fish. Current knowledge of these worms is for the most part limited to their morphological, phylogenetic, and population features. Information concerning the biochemical and molecular nature of physiological processes involved in host-parasite interaction, such as evasion of the immune system and its regulation, digestion of macromolecules, suppression of blood coagulation and inflammation, and effect on host tissue and physiology, is lacking. In this study, we report for the first time a comprehensive transcriptomic/secretome description of expressed genes and proteins secreted by the adult stage of Eudiplozoon nipponicum (Goto, 1891) Khotenovsky, 1985, an obligate sanguivorous monogenean which parasitises the gills of the common carp (Cyprinus carpio). RESULTS: RNA-seq raw reads (324,941 Roche 454 and 149,697,864 Illumina) were generated, de novo assembled, and filtered into 37,062 protein-coding transcripts. For 19,644 (53.0%) of them, we determined their sequential homologues. In silico functional analysis of E. nipponicum RNA-seq data revealed numerous transcripts, pathways, and GO terms responsible for immunomodulation (inhibitors of proteolytic enzymes, CD59-like proteins, fatty acid binding proteins), feeding (proteolytic enzymes cathepsins B, D, L1, and L3), and development (fructose 1,6-bisphosphatase, ferritin, and annexin). LC-MS/MS spectrometry analysis identified 721 proteins secreted by E. nipponicum with predominantly immunomodulatory and anti-inflammatory functions (peptidyl-prolyl cis-trans isomerase, homolog to SmKK7, tetraspanin) and ability to digest host macromolecules (cathepsins B, D, L1). CONCLUSIONS: In this study, we integrated two high-throughput sequencing techniques, mass spectrometry analysis, and comprehensive bioinformatics approach in order to arrive at the first comprehensive description of monogenean transcriptome and secretome. Exploration of E. nipponicum transcriptome-related nucleotide sequences and translated and secreted proteins offer a better understanding of molecular biology and biochemistry of these, often neglected, organisms. It enabled us to report the essential physiological pathways and protein molecules involved in their interactions with the fish hosts.

• MeSH
• anotace sekvence MeSH
• chromatografie kapalinová MeSH
• fylogeneze MeSH
• kapři * genetika   MeSH
• stanovení celkové genové exprese MeSH
• tandemová hmotnostní spektrometrie MeSH
• transkriptom MeSH
• Trematoda * genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The gills of the common carp, whose mucosal surface belongs to the key defence mechanisms of piscine immunity, can be infested with both the larval and adult stage of Eudiplozoon nipponicum (Monogenea). Although on their own, monogeneans do not considerably compromise their hosts' health status, fish with epithelial barriers damaged in parasite feeding and attachment sites are at an increased risk of bacterial challenge with possible harmful consequences. Several studies suggest that helminth parasites of teleost fish evade and manipulate host immune system via their excretory-secretory products, but our knowledge of these processes in the monogeneans is limited. Cysteine peptidase inhibitors (CPI), which are found in the secretions of numerous parasites, often induce immunosuppression by subverting Th1 mechanisms and drawing the immune system towards a Th2/Treg response. We employed the qPCR to test the effect of recently characterised CPI of E. nipponicum (rEnStef) on the mRNA expression of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 produced by porcine macrophages in vitro. After an initial preincubation with rEnStef, we stimulated the macrophages using LPS. By inducing a Th1 pro-inflammatory response, we imitated the immune reaction during a bacterial challenge in tissue damaged by the feeding and attachment of E. nipponicum. We observed a significant dose-dependent downregulation of the expression of TNF-α and IL-10 cytokines. The observed suppression of TNF-alpha expression by rEnStef could result in decreased pathogen control, which might in turn lead to increased rates of secondary bacterial infections in fish infected by E. nipponicum.

• MeSH
• cytokiny * účinky léků   metabolismus   MeSH
• imunomodulace MeSH
• inhibitory cysteinových proteinas farmakologie   MeSH
• interleukin-10 metabolismus   MeSH
• kapři parazitologie   MeSH
• makrofágy * účinky léků   metabolismus   MeSH
• prasata MeSH
• rekombinantní proteiny farmakologie   MeSH
• TNF-alfa účinky léků   metabolismus   MeSH
• Trematoda imunologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Eudiplozoon nipponicum (Goto, 1891) is a hematophagous monogenean ectoparasite which inhabits the gills of the common carp (Cyprinus carpio). Heavy infestation can lead to anemia and in conjunction with secondary bacterial infections cause poor health and eventual death of the host. This study is based on an innovative approach to protein localization which has never been used in parasitology before. Using laser capture microdissection, we dissected particular areas of the parasite body without contaminating the samples by surrounding tissue and in combination with analysis by mass spectrometry obtained tissue-specific proteomes of tegument, intestine, and parenchyma of our model organism, E. nipponicum. We successfully verified the presence of certain functional proteins (e.g. cathepsin L) in tissues where their presence was expected (intestine) and confirmed that there were no traces of these proteins in other tissues (tegument and parenchyma). Additionally, we identified a total of 2,059 proteins, including 72 peptidases and 33 peptidase inhibitors. As expected, the greatest variety was found in the intestine and the lowest variety in the parenchyma. Our results are significant on two levels. Firstly, we demonstrated that one can localize all proteins in one analysis and without using laboratory animals (antibodies for immunolocalization of single proteins). Secondly, this study offers the first complex proteomic data on not only the E. nipponicum but within the whole class of Monogenea, which was from this point of view until recently neglected.

• MeSH
• kapři parazitologie   MeSH
• kathepsiny analýza   metabolismus   MeSH
• laserová záchytná mikrodisekce MeSH
• parenchymatická tkáň metabolismus   MeSH
• ploštěnci metabolismus   MeSH
• proteasy analýza   metabolismus   MeSH
• proteom analýza   MeSH
• proteomika metody   MeSH
• střevní sliznice metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• žábry parazitologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Serine peptidases are involved in many physiological processes including digestion, haemostasis and complement cascade. Parasites regulate activities of host serine peptidases to their own benefit, employing various inhibitors, many of which belong to the Kunitz-type protein family. In this study, we confirmed the presence of potential anticoagulants in protein extracts of the haematophagous monogenean Eudiplozoon nipponicum which parasitizes the common carp. We then focused on a Kunitz protein (EnKT1) discovered in the E. nipponicum transcriptome, which structurally resembles textilinin-1, an antihemorrhagic snake venom factor from Pseudonaja textilis. The protein was recombinantly expressed, purified and biochemically characterised. The recombinant EnKT1 did inhibit in vitro activity of Factor Xa of the coagulation cascade, but exhibited a higher activity against plasmin and plasma kallikrein, which participate in fibrinolysis, production of kinins, and complement activation. Anti-coagulation properties of EnKT1 based on the inhibition of Factor Xa were confirmed by thromboelastography, but no effect on fibrinolysis was observed. Moreover, we discovered that EnKT1 significantly impairs the function of fish complement, possibly by inhibiting plasmin or Factor Xa which can act as a C3 and C5 convertase. We localised Enkt1 transcripts and protein within haematin digestive cells of the parasite by RNA in situ hybridisation and immunohistochemistry, respectively. Based on these results, we suggest that the secretory Kunitz protein of E. nipponicum has a dual function. In particular, it impairs both haemostasis and complement activation in vitro, and thus might facilitate digestion of a host's blood and protect a parasite's gastrodermis from damage by the complement. This study presents, to our knowledge, the first characterisation of a Kunitz protein from monogeneans and the first example of a parasite Kunitz inhibitor that impairs the function of the complement.

• MeSH
• antifibrinolytika chemie   imunologie   MeSH
• antikoagulancia chemie   imunologie   MeSH
• faktor Xa imunologie   MeSH
• hemostáza * MeSH
• infekce červy třídy Trematoda krev   imunologie   parazitologie   veterinární   MeSH
• inhibitory enzymů chemie   imunologie   MeSH
• inhibitory faktoru Xa chemie   imunologie   MeSH
• interakce hostitele a parazita MeSH
• kapři krev   imunologie   parazitologie   MeSH
• komplement imunologie   MeSH
• nemoci ryb krev   imunologie   parazitologie   MeSH
• plasmin imunologie   MeSH
• plazmatický kalikrein antagonisté a inhibitory   imunologie   MeSH
• proteiny červů chemie   genetika   imunologie   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• Trematoda chemie   genetika   imunologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• biogenní aminy analýza   MeSH
• imunohistochemie MeSH
• nervový systém chemie   ultrastruktura   MeSH
• Trematoda ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH