Dermanyssus gallinae is a blood-feeding mite that parasitises wild birds and farmed poultry. Its remarkably swift processing of blood, together with the capacity to blood-feed during most developmental stages, makes this mite a highly debilitating pest. To identify specific adaptations to digestion of a haemoglobin-rich diet, we constructed and compared transcriptomes from starved and blood-fed stages of the parasite and identified midgut-enriched transcripts. We noted that midgut transcripts encoding cysteine proteases were upregulated with a blood meal. Mapping the full proteolytic apparatus, we noted a reduction in the suite of cysteine proteases, missing homologues for Cathepsin B and C. We have further identified and phylogenetically analysed three distinct transcripts encoding vitellogenins that facilitate the reproductive capacity of the mites. We also fully mapped transcripts for haem biosynthesis and the ferritin-based system of iron storage and inter-tissue trafficking. Additionally, we identified transcripts encoding proteins implicated in immune signalling (Toll and IMD pathways) and activity (defensins and thioester-containing proteins), RNAi, and ion channelling (with targets for commercial acaricides such as Fluralaner, Fipronil, and Ivermectin). Viral sequences were filtered from the Illumina reads and we described, in part, the RNA-virome of D. gallinae with identification of a novel virus, Red mite quaranjavirus 1.

• MeSH
• drůbež MeSH
• infestace roztoči * veterinární   parazitologie   MeSH
• kur domácí MeSH
• nemoci drůbeže * MeSH
• roztoči * genetika   MeSH
• sekvenování transkriptomu MeSH
• virom MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH

The hard tick Ixodes ricinus is a vector of Lyme disease and tick-borne encephalitis. Host blood protein digestion, essential for tick development and reproduction, occurs in tick midgut digestive cells driven by cathepsin proteases. Little is known about the regulation of the digestive proteolytic machinery of I. ricinus. Here we characterize a novel cystatin-type protease inhibitor, mialostatin, from the I. ricinus midgut. Blood feeding rapidly induced mialostatin expression in the gut, which continued after tick detachment. Recombinant mialostatin inhibited a number of I. ricinus digestive cysteine cathepsins, with the greatest potency observed against cathepsin L isoforms, with which it co-localized in midgut digestive cells. The crystal structure of mialostatin was determined at 1.55 Å to explain its unique inhibitory specificity. Finally, mialostatin effectively blocked in vitro proteolysis of blood proteins by midgut cysteine cathepsins. Mialostatin is likely to be involved in the regulation of gut-associated proteolytic pathways, making midgut cystatins promising targets for tick control strategies.

• Klíčová slova
• Ixodes ricinus, cathepsin, crystal structure, cysteine protease, digestion, midgut, parasite,
• MeSH
• cystatiny metabolismus   MeSH
• fylogeneze MeSH
• kathepsin L metabolismus   MeSH
• klíšťata metabolismus   MeSH
• klíště metabolismus   MeSH
• krevní proteiny metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• proteolýza MeSH
• sekvence aminokyselin MeSH
• trávicí systém metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cystatiny MeSH
• kathepsin L MeSH
• krevní proteiny 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.

• Klíčová slova
• Blood digestion, Cathepsin, Cysteine peptidase, Diplozoidae, Eudiplozoon nipponicum, Fish parasite, Haematophagy, Monogenea, Protease, S2 subsite,
• 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
• Názvy látek
• kathepsin B MeSH
• kathepsin L MeSH
• rekombinantní proteiny MeSH

Adult females of the genus Ixodes imbibe blood meals exceeding about 100 times their own weight within 7‒9 days. During this period, ticks internalise components of host blood by endocytic digest cells that line the tick midgut epithelium. Using RNA-seq, we aimed to characterise the midgut transcriptome composition in adult Ixodes ricinus females during early and late phase of engorgement. To address specific adaptations to the haemoglobin-rich diet, we compared the midgut transcriptomes of genetically homogenous female siblings fed either bovine blood or haemoglobin-depleted serum. We noted that tick gut transcriptomes are subject to substantial temporal-dependent expression changes between day 3 and day 8 of feeding. In contrast, the number of transcripts significantly affected by the presence or absence of host red blood cells was low. Transcripts relevant to the processes associated with blood-meal digestion were analysed and involvement of selected encoded proteins in the tick midgut physiology discussed. A total of 7215 novel sequences from I. ricinus were deposited in public databases as an additional outcome of this study. Our results broaden the current knowledge of tick digestive system and may lead to the discovery of potential molecular targets for efficient tick control.

• MeSH
• klíště genetika   metabolismus   MeSH
• sekvenční analýza RNA * MeSH
• skot MeSH
• stanovení celkové genové exprese * MeSH
• střeva patologie   MeSH
• střevní sliznice metabolismus   MeSH
• transkriptom fyziologie   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH

To identify the gut-associated tick aspartic hemoglobinase, this work focuses on the functional diversity of multiple Ixodes ricinus cathepsin D forms (IrCDs). Out of three encoding genes representing Ixodes scapularis genome paralogs, IrCD1 is the most distinct enzyme with a shortened propeptide region and a unique pattern of predicted post-translational modifications. IrCD1 gene transcription is induced by tick feeding and is restricted to the gut tissue. The hemoglobinolytic role of IrCD1 was further supported by immunolocalization of IrCD1 in the vesicles of tick gut cells. Properties of recombinantly expressed rIrCD1 are consistent with the endo-lysosomal environment because the zymogen is autoactivated and remains optimally active in acidic conditions. Hemoglobin cleavage pattern of rIrCD1 is identical to that produced by the native enzyme. The preference for hydrophobic residues at the P1 and P1' position was confirmed by screening a novel synthetic tetradecapeptidyl substrate library. Outside the S1-S1' regions, rIrCD1 tolerates most amino acids but displays a preference for tyrosine at P3 and alanine at P2'. Further analysis of the cleavage site location within the peptide substrate indicated that IrCD1 is a true endopeptidase. The role in hemoglobinolysis was verified with RNAi knockdown of IrCD1 that decreased gut extract cathepsin D activity by >90%. IrCD1 was newly characterized as a unique hemoglobinolytic cathepsin D contributing to the complex intestinal proteolytic network of mainly cysteine peptidases in ticks.

• MeSH
• genetická transkripce fyziologie   MeSH
• genom fyziologie   MeSH
• hemoglobiny genetika   metabolismus   MeSH
• kathepsin D genetika   metabolismus   MeSH
• klíště enzymologie   genetika   MeSH
• posttranslační úpravy proteinů fyziologie   MeSH
• proteiny členovců genetika   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• střeva enzymologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hemoglobiny MeSH
• kathepsin D MeSH
• proteiny členovců MeSH
• rekombinantní proteiny MeSH