Trematodes of the order Diplostomida are well known as serious pathogens of man, and both farm and wild animals; members of the genus Schistosoma (Schistosomatidae) are responsible for human schistosomosis (schistosomiasis) affecting more than 200 million people in tropical and subtropical countries, and infections of mammals and birds by animal schistosomes are of great veterinary importance. The order Diplostomida is also rich in species parasitizing other major taxa of vertebrates. The "Aporocotylidae" sensu lato are pathogenic in fish, "Spirorchiidae" sensu lato in reptiles. All these flukes have two-host life cycles, with asexually reproducing larvae usually in mollusks and occasionally in annelids, and adults usually live in the blood vessels of their vertebrate hosts. Pathology is frequently associated with inflammatory reactions to eggs trapped in various tissues/organs. On the other hand, the representatives of Diplostomidae and Strigeidae have three- or four-host life cycles in which vertebrates often serve not only as definitive but also as intermediate or paratenic hosts. Pathology is usually associated with migration of metacercariae and mesocercariae within the host tissues. The impact of these trematode infections on both farm and wild animals may be significant.
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.
Antibody trapping is a recently described strategy for immune evasion observed in the intestinal trematode Echinostoma caproni, which may aid to avoiding the host humoral response, thus facilitating parasite survival in the presence of high levels of local-specific antibodies. Parasite-derived peptidases carry out the degradation of trapped antibodies, being essential for this mechanism. Herein, we show that cathepsin-like cysteine endopeptidases are active in the excretory/secretory products (ESPs) of E. caproni and play an important role in the context of antibody trapping. Cysteine endopeptidase activity was detected in the ESPs of E. caproni adults. The affinity probe DCG-04 distinguished a cysteine peptidase band in ESPs, which was specifically recognized by an anti-cathepsin L heterologous antibody. The same antibody localized this protein in the gut and syncytial tegument of adult worms. Studies with cultured parasites showed that in vivo-bound antibodies are removed from the parasite surface in the absence of peptidase inhibitors, while addition of cathepsin L inhibitor prevented their degradation. These results indicate that cathepsin L-like peptidases are involved in the degradation of surface-trapped antibodies and suggest that cysteine peptidases are not only crucial for tissue-invading trematodes, but they can be equally relevant at the parasite-host interface in gut-dwelling flukes.
- MeSH
- cysteinové endopeptidasy metabolismus MeSH
- Echinostoma imunologie metabolismus MeSH
- echinostomióza imunologie parazitologie MeSH
- imunitní únik imunologie MeSH
- kathepsin L antagonisté a inhibitory MeSH
- proteolýza MeSH
- protilátky protozoální imunologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Trematodes of the order Diplostomida are well known as serious pathogens of man, and both farm and wild animals; members of the genus Schistosoma (Schistosomatidae) are responsible for human schistosomosis affecting more than 200 million people in tropical and subtropical countries, infections of mammals and birds by animal schistosomes are of great veterinary importance. The order Diplostomida is also rich in species parasitizing other major taxa of vertebrates. The Aporocotylidae are pathogenic in fish, Spirorchiidae in reptiles. All these flukes have two-host life cycles, with asexually reproducing larvae usually in molluscs and occasionally in annelids, and adults usually live in the blood vessels of their vertebrate hosts. Pathology is frequently associated with inflammatory reactions to eggs trapped in various tissues/organs. On the other hand, the representatives of Diplostomidae and Strigeidae have three- or four-host life cycles in which vertebrates often serve not only as definitive, but also as intermediate or paratenic hosts. Pathology is usually associated with migration of metacercariae and mesocercariae within the host tissues. The impact of these trematode infections on both farm and wild animals may be significant.
- MeSH
- infekce červy třídy Trematoda * parazitologie přenos MeSH
- interakce hostitele a parazita MeSH
- lidé MeSH
- měkkýši parazitologie MeSH
- nemoci ryb parazitologie MeSH
- ryby parazitologie MeSH
- stadia vývoje MeSH
- Trematoda * fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články 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
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
Cercarial dermatitis (CD) is an allergic skin disease that rises in consequence of infection by invasive stages (cercariae) of trematodes of the family Schistosomatidae. CD has been considered a re-emerging disease, human cases have been reported from all continents, and tourism-threatening outbreaks occur even in frequented recreational areas. Although the symptoms of CD are generally known, the data on immune response in human patients are sporadic and incomprehensive. In the present study, we attempted to correlate the symptoms, personal history, and time course of CD in human patients with differential cell counts, dynamics of selected cytokines, and dynamics and quality of antibody response. By a systematic follow-up, we obtained a uniquely complex dataset from ten persons accidentally and concurrently infected by the same parasite species in the same locality. The onset of CD was significantly faster, and the symptoms were heavier in participants with a history of CD if compared to naive ones, who, however, also developed some of the symptoms. The repeatedly infected persons had elevated proportion of eosinophils 1 week post exposure (p.e.) and a stronger specific IgG but not IgM response, whereas specific IgE response was not observed. Increased serum levels of IL-4 occurred 1 and 3 week(s) p.e. in all participants. There was high variability in individual immunoblot patterns of IgG response, and no antigen with a universal diagnostic potential was confirmed. The presented analyses suggested that a complex approach can improve the accuracy of the diagnosis of CD, but component data should be interpreted carefully.
- MeSH
- dermatitida imunologie parazitologie MeSH
- dospělí MeSH
- epidemický výskyt choroby MeSH
- imunoglobulin E krev MeSH
- imunoglobulin G krev MeSH
- imunoglobulin M krev MeSH
- infekce červy třídy Trematoda diagnóza imunologie parazitologie MeSH
- interleukin-4 krev MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- následné studie MeSH
- protilátky protozoální krev MeSH
- průzkumy a dotazníky MeSH
- rybníky parazitologie MeSH
- Schistosomatidae imunologie MeSH
- zvířata MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
- Klíčová slova
- Taenia crassiceps,
- MeSH
- cysticerkóza * chirurgie diagnóza genetika parazitologie veterinární MeSH
- fatální výsledek MeSH
- Galagidae parazitologie MeSH
- nemoci psů * MeSH
- neurocysticercosis etiologie MeSH
- psi parazitologie MeSH
- sekvenční analýza DNA veterinární MeSH
- syndromy imunologické nedostatečnosti parazitologie MeSH
- Taenia * parazitologie patogenita růst a vývoj MeSH
- tenióza * diagnóza genetika parazitologie veterinární MeSH
- zvířata MeSH
- Check Tag
- psi parazitologie MeSH
- zvířata MeSH
- Publikační typ
- kazuistiky MeSH
- Geografické názvy
- Česká republika MeSH
BACKGROUND: Cysteine peptidases of clan CA, family C1 account for a major part of proteolytic activity in the haematophagous monogenean Eudiplozoon nipponicum. The full spectrum of cysteine cathepsins is, however, unknown and their particular biochemical properties, tissue localisation, and involvement in parasite-host relationships are yet to be explored. METHODS: Sequences of cathepsins L and B (EnCL and EnCB) were mined from E. nipponicum transcriptome and analysed bioinformatically. Genes encoding two EnCLs and one EnCB were cloned and recombinant proteins produced in vitro. The enzymes were purified by chromatography and their activity towards selected substrates was characterised. Antibodies and specific RNA probes were employed for localisation of the enzymes/transcripts in tissues of E. nipponicum adults. RESULTS: Transcriptomic analysis revealed a set of ten distinct transcripts that encode EnCLs. The enzymes are significantly variable in their active sites, specifically the S2 subsites responsible for interaction with substrates. Some of them display unusual structural features that resemble cathepsins B and S. Two recombinant EnCLs had different pH activity profiles against both synthetic and macromolecular substrates, and were able to hydrolyse blood proteins and collagen I. They were localised in the haematin cells of the worm's digestive tract and in gut lumen. The EnCB showed similarity with cathepsin B2 of Schistosoma mansoni. It displays molecular features typical of cathepsins B, including an occluding loop responsible for its exopeptidase activity. Although the EnCB hydrolysed haemoglobin in vitro, it was localised in the vitelline cells of the parasite and not the digestive tract. CONCLUSIONS: To our knowledge, this study represents the first complex bioinformatic and biochemical characterisation of cysteine peptidases in a monogenean. Eudiplozoon nipponicum adults express a variety of CLs, which are the most abundant peptidases in the worms. The properties and localisation of the two heterologously expressed EnCLs indicate a central role in the (partially extracellular?) digestion of host blood proteins. High variability of substrate-binding sites in the set of EnCLs suggests specific adaptation to a range of biological processes that require proteolysis. Surprisingly, a single cathepsin B is expressed by the parasite and it is not involved in digestion, but probably in vitellogenesis.
- MeSH
- gastrointestinální trakt parazitologie MeSH
- hydrolýza MeSH
- interakce hostitele a parazita MeSH
- kapři parazitologie MeSH
- kathepsin B chemie genetika izolace a purifikace metabolismus MeSH
- kathepsin L chemie genetika izolace a purifikace metabolismus MeSH
- proteolýza MeSH
- rekombinantní proteiny analýza genetika izolace a purifikace MeSH
- stanovení celkové genové exprese MeSH
- Trematoda enzymologie genetika MeSH
- zavlečené druhy MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Klíčová slova
- Pyrvinium,
- MeSH
- anthelmintika MeSH
- dítě MeSH
- dospělí MeSH
- enterobióza * diagnóza epidemiologie farmakoterapie přenos MeSH
- Enterobius patogenita MeSH
- environmentální zdraví MeSH
- kojení MeSH
- komplikace těhotenství MeSH
- lidé MeSH
- mebendazol aplikace a dávkování farmakokinetika terapeutické užití MeSH
- příznaky a symptomy MeSH
- pyrviniové sloučeniny terapeutické užití zásobování a distribuce MeSH
- těhotenství MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Geografické názvy
- Česká republika MeSH
