BACKGROUND: The blood flukes of genus Schistosoma are the causative agent of schistosomiasis, a parasitic disease that infects more than 200 million people worldwide. Proteases of schistosomes are involved in critical steps of host-parasite interactions and are promising therapeutic targets. We recently identified and characterized a group of S1 family Schistosoma mansoni serine proteases, including SmSP1 to SmSP5. Expression levels of some SmSPs in S. mansoni are low, and by standard genome sequencing technologies they are marginally detectable at the method threshold levels. Here, we report their spatial gene expression patterns in adult S. mansoni by the high-sensitivity localization assay. METHODOLOGY: Highly sensitive fluorescence in situ RNA hybridization (FISH) was modified and used for the localization of mRNAs encoding individual SmSP proteases (including low-expressed SmSPs) in tissues of adult worms. High sensitivity was obtained due to specifically prepared tissue and probes in combination with the employment of a signal amplification approach. The assay method was validated by detecting the expression patterns of a set of relevant reference genes including SmCB1, SmPOP, SmTSP-2, and Sm29 with localization formerly determined by other techniques. RESULTS: FISH analysis revealed interesting expression patterns of SmSPs distributed in multiple tissues of S. mansoni adults. The expression patterns of individual SmSPs were distinct but in part overlapping and were consistent with existing transcriptome sequencing data. The exception were genes with significantly low expression, which were also localized in tissues where they had not previously been detected by RNA sequencing methods. In general, SmSPs were found in various tissues including reproductive organs, parenchymal cells, esophagus, and the tegumental surface. CONCLUSIONS: The FISH-based assay provided spatial information about the expression of five SmSPs in adult S. mansoni females and males. This highly sensitive method allowed visualization of low-abundantly expressed genes that are below the detection limits of standard in situ hybridization or by RNA sequencing. Thus, this technical approach turned out to be suitable for sensitive localization studies and may also be applicable for other trematodes. The results suggest that SmSPs may play roles in diverse processes of the parasite. Certain SmSPs expressed at the surface may be involved in host-parasite interactions.

• MeSH
• exprese genu * MeSH
• hybridizace in situ fluorescenční metody   normy   MeSH
• proteiny červů genetika   MeSH
• RNA metabolismus   MeSH
• Schistosoma mansoni enzymologie   genetika   MeSH
• serinové proteasy genetika   MeSH
• stanovení celkové genové exprese MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví 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

In freshwater ecosystems, snails can significantly influence the competition between primary producers through grazing of periphyton. This activity can potentially be modified by trematodes, a large group of parasites which mostly use molluscs as the first intermediate host. Available studies, however, show contradictory effects of trematodes on snail periphyton grazing. Here, we used four different freshwater snail-trematode systems to test whether a general pattern can be detected for the impact of trematode infections on snail periphyton grazing. In our experimental systems, mass-specific periphyton grazing rates of infected snails were higher, lower, or similar to rates of non-infected conspecifics, suggesting that no general pattern exists. The variation across studied snail-trematode systems may result from differences on how the parasite uses the resources of the snail and thus affects their energy budget. Trematode infections can significantly alter the grazing rate of snails, where, depending on the system, the mass-specific grazing rate can double or halve. This underlines both, the high ecological relevance of trematodes and the need for comprehensive studies at the species level to allow an integration of these parasite-host interactions into aquatic food web concepts.

• MeSH
• ekologie MeSH
• ekosystém MeSH
• hlemýždi parazitologie   MeSH
• infekce červy třídy Trematoda parazitologie   MeSH
• interakce hostitele a parazita fyziologie   MeSH
• perifyton * MeSH
• potravní řetězec MeSH
• přijímání potravy fyziologie   MeSH
• sladká voda parazitologie   MeSH
• Trematoda izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články 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