BACKGROUND: Ticks, hematophagous Acari, pose a significant threat by transmitting various pathogens to their vertebrate hosts during feeding. Despite advances in tick genomics, high-quality genomes were lacking until recently, particularly in the genus Ixodes, which includes the main vectors of Lyme disease. RESULTS: Here, we present the genome sequences of four tick species, derived from a single female individual, with a particular focus on the European species Ixodes ricinus, achieving a chromosome-level assembly. Additionally, draft assemblies were generated for the three other Ixodes species, I. persulcatus, I. pacificus, and I. hexagonus. The quality of the four genomes and extensive annotation of several important gene families have allowed us to study the evolution of gene repertoires at the level of the genus Ixodes and of the tick group. We have determined gene families that have undergone major amplifications during the evolution of ticks, while an expression atlas obtained for I. ricinus reveals striking patterns of specialization both between and within gene families. Notably, several gene family amplifications are associated with a proliferation of single-exon genes-most strikingly for fatty acid elongases and sulfotransferases. CONCLUSIONS: The integration of our data with existing genomes establishes a solid framework for the study of gene evolution, improving our understanding of tick biology. In addition, our work lays the foundations for applied research and innovative control targeting these organisms.

• Klíčová slova
• Comparative genomics, Duplication, Hematophagy, Parasite, Retroposition,
• MeSH
• genom * MeSH
• klíště * genetika   MeSH
• molekulární evoluce MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Tick-borne encephalitis virus (TBEV) is flavivirus transmitted to the host via tick saliva which contains various molecules with biological impacts. One of such molecules is Iristatin, a cysteine protease inhibitor from Ixodes ricinus that has been shown to have immunomodulatory properties. To characterize Iristatin in the relation to TBEV, we investigate whether this tick inhibitor has any capacity to influence TBEV infection. Mice were intradermally infected by TBEV with or without Iristatin and the viral multiplication was determined in skin and brain tissues by RT-PCR two and 5 days after infection. The viral RNA was detected in both intervals in skin and increased by time. The application of Iristatin caused a reduction in viral RNA in skin but not in the brain of infected mice 5 days post-infection. Moreover, anti-viral effect of Iristatin on skin was accompanied by a significant decline of interferon-stimulated gene 15 gene expression. The effect of Iristatin on TBEV replication was tested also in vitro in primary macrophages and dendritic cells; however, no changes were observed suggesting no direct interference of Iristatin with virus replication. Still, the Iristatin caused a suppression of Erk1/2 phosphorylation in TBEV-infected dendritic cells and had the anti-apoptotic effect. This is the first report showing that a tick cystatin decreases the viral RNA in the host skin, likely indirectly through creating skin environment that is less supportive for TBEV replication. Assuming, that viral RNA reflects the amount of infectious virus, decline of TBEV in host skin could influence the tick biology or virus transmission during cofeeding.

• Klíčová slova
• Cystatin, Flavivirus, Tick, Tick-borne encephalitis virus, Virus replication,
• MeSH
• antivirové látky farmakologie   MeSH
• cystatiny farmakologie   metabolismus   genetika   MeSH
• dendritické buňky virologie   účinky léků   MeSH
• klíště * virologie   účinky léků   MeSH
• klíšťová encefalitida * virologie   MeSH
• kůže * virologie   MeSH
• makrofágy virologie   MeSH
• mozek virologie   metabolismus   MeSH
• myši MeSH
• replikace viru * účinky léků   MeSH
• RNA virová genetika   MeSH
• slinné cystatiny metabolismus   MeSH
• viry klíšťové encefalitidy * účinky léků   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antivirové látky MeSH
• cystatiny MeSH
• RNA virová MeSH
• slinné cystatiny MeSH

Protease inhibitors regulate various biological processes and prevent host tissue/organ damage. Specific inhibition/regulation of proteases is clinically valuable for treating several diseases. Psoriasis affects the skin in the limbs and scalp of the body, and the contribution of cysteine and serine proteases to the development of skin inflammation is well documented. Cysteine protease inhibitors from ticks have high specificity, selectivity, and affinity to their target proteases and are efficient immunomodulators. However, their potential therapeutic effect on psoriasis pathogenesis remains to be determined. Therefore, we tested four tick cystatins (Sialostatin L, Sialostatin L2, Iristatin, and Mialostatin) in the recently developed, innate immunity-dependent mannan-induced psoriasis model. We explored the effects of protease inhibitors on clinical symptoms and histological features. In addition, the number and percentage of immune cells (dendritic cells, neutrophils, macrophages, and γδT cells) by flow cytometry, immunofluorescence/immunohistochemistry and, the expression of pro-inflammatory cytokines (TNF-a, IL-6, IL-22, IL-23, and IL-17 family) by qPCR were analyzed using skin, spleen, and lymph node samples. Tick protease inhibitors have significantly decreased psoriasis symptoms and disease manifestations but had differential effects on inflammatory responses and immune cell populations, suggesting different modes of action of these inhibitors on psoriasis-like inflammation. Thus, our study demonstrates, for the first time, the usefulness of tick-derived protease inhibitors for treating skin inflammation in patients.

• Klíčová slova
• autoimmune disease, immune responses, protease inhibitors, psoriasis, tick,
• MeSH
• dermatitida * MeSH
• endopeptidasy MeSH
• inhibitory cysteinových proteinas MeSH
• inhibitory proteas MeSH
• lidé MeSH
• mannany MeSH
• přirozená imunita MeSH
• proteasy MeSH
• psoriáza * chemicky indukované   farmakoterapie   MeSH
• zánět farmakoterapie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• endopeptidasy MeSH
• inhibitory cysteinových proteinas MeSH
• inhibitory proteas MeSH
• mannany MeSH
• proteasy MeSH

The structure and biochemical properties of protease inhibitors from the thyropin family are poorly understood in parasites and pathogens. Here, we introduce a novel family member, Ir-thyropin (IrThy), which is secreted in the saliva of Ixodes ricinus ticks, vectors of Lyme borreliosis and tick-borne encephalitis. The IrThy molecule consists of two consecutive thyroglobulin type-1 (Tg1) domains with an unusual disulfide pattern. Recombinant IrThy was found to inhibit human host-derived cathepsin proteases with a high specificity for cathepsins V, K, and L among a wide range of screened cathepsins exhibiting diverse endo- and exopeptidase activities. Both Tg1 domains displayed inhibitory activities, but with distinct specificity profiles. We determined the spatial structure of one of the Tg1 domains by solution NMR spectroscopy and described its reactive center to elucidate the unique inhibitory specificity. Furthermore, we found that the inhibitory potency of IrThy was modulated in a complex manner by various glycosaminoglycans from host tissues. IrThy was additionally regulated by pH and proteolytic degradation. This study provides a comprehensive structure-function characterization of IrThy-the first investigated thyropin of parasite origin-and suggests its potential role in host-parasite interactions at the tick bite site.

• Klíčová slova
• cathepsin, cysteine protease, parasite, protease inhibitor, protein structure, saliva, thyropin, tick,
• MeSH
• cystein MeSH
• glykosaminoglykany MeSH
• kathepsiny metabolismus   MeSH
• klíště * metabolismus   MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• sliny * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cystein MeSH
• glykosaminoglykany MeSH
• kathepsiny MeSH

Ticks are ectoparasites that feed on blood and have an impressive ability to consume and process enormous amounts of host blood, allowing extremely long periods of starvation between blood meals. The central role in the parasitic lifestyle of ticks is played by the midgut. This organ efficiently stores and digests ingested blood and serves as the primary interface for the transmission of tick-borne pathogens. In this study, we used a label-free quantitative approach to perform a novel dynamic proteomic analysis of the midgut of Ixodesricinus nymphs, covering their development from unfed to pre-molt stages. We identified 1534 I. ricinus-specific proteins with a relatively low proportion of host proteins. This proteome dataset, which was carefully examined by manual scrutiny, allowed precise annotation of proteins important for blood meal processing and their dynamic changes during nymphal ontogeny. We focused on midgut molecules related to lipid hydrolysis, storage, and transport, opening a yet unexplored avenue for studying lipid metabolism in ticks. Further dynamic profiling of the tick's multi-enzyme digestive network, protease inhibitors, enzymes involved in redox homeostasis and detoxification, antimicrobial peptides, and proteins responsible for midgut colonization by Borrelia spirochetes promises to uncover new targets for targeting tick nymphs, the most critical life stage for transmission the pathogens that cause tick-borne diseases.

• Klíčová slova
• Borrelia, Ixodes, antimicrobial peptides, label-free quantification, lipid metabolism, midgut, protease inhibitors, proteases, proteome, ticks,
• MeSH
• klíště * parazitologie   MeSH
• proteom MeSH
• proteomika MeSH
• trávicí systém MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteom MeSH

Tick saliva injected into the vertebrate host contains bioactive anti-proteolytic proteins from the cystatin family; however, the molecular basis of their unusual biochemical and physiological properties, distinct from those of host homologs, is unknown. Here, we present Ricistatin, a novel secreted cystatin identified in the salivary gland transcriptome of Ixodes ricinus ticks. Recombinant Ricistatin inhibited host-derived cysteine cathepsins and preferentially targeted endopeptidases, while having only limited impact on proteolysis driven by exopeptidases. Determination of the crystal structure of Ricistatin in complex with a cysteine cathepsin together with characterization of structural determinants in the Ricistatin binding site explained its restricted specificity. Furthermore, Ricistatin was potently immunosuppressive and anti-inflammatory, reducing levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α and nitric oxide in macrophages; IL-2 and IL-9 levels in Th9 cells; and OVA antigen-induced CD4+ T cell proliferation and neutrophil migration. This work highlights the immunotherapeutic potential of Ricistatin and, for the first time, provides structural insights into the unique narrow selectivity of tick salivary cystatins determining their bioactivity.

• Klíčová slova
• Cystatins, Host–parasite interactions, Ixodes ricinus, Protease inhibition, Protein structure, Tick saliva,
• MeSH
• cystatiny * farmakologie   MeSH
• cystein metabolismus   MeSH
• endopeptidasy metabolismus   MeSH
• kathepsiny metabolismus   MeSH
• klíště * chemie   MeSH
• obratlovci MeSH
• proteasy metabolismus   MeSH
• slinné cystatiny chemie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cystatiny * MeSH
• cystein MeSH
• endopeptidasy MeSH
• kathepsiny MeSH
• proteasy MeSH
• slinné cystatiny MeSH

Fasciolosis is a worldwide parasitic disease of ruminants and an emerging human disease caused by the liver fluke Fasciola hepatica. The cystatin superfamily of cysteine protease inhibitors is composed of distinct families of intracellular stefins and secreted true cystatins. FhCyLS-2 from F. hepatica is an unusual member of the superfamily, where our sequence and 3D structure analyses in this study revealed that it combines characteristics of both families. The protein architecture demonstrates its relationship to stefins, but FhCyLS-2 also contains the secretion signal peptide and disulfide bridges typical of true cystatins. The secretion status was confirmed by detecting the presence of FhCyLS-2 in excretory/secretory products, supported by immunolocalization. Our high-resolution crystal structure of FhCyLS-2 showed a distinct disulfide bridging pattern and functional reactive center. We determined that FhCyLS-2 is a broad specificity inhibitor of cysteine cathepsins from both the host and F. hepatica, suggesting a dual role in the regulation of exogenous and endogenous proteolysis. Based on phylogenetic analysis that identified several FhCyLS-2 homologues in liver/intestinal foodborne flukes, we propose a new group within the cystatin superfamily called cystatin-like stefins.

• Klíčová slova
• cystatin, cysteine cathepsin, helminth parasite, protease inhibitor, protein evolution, protein structure, stefin,
• MeSH
• cystatiny * genetika   chemie   MeSH
• disulfidy MeSH
• Fasciola hepatica * genetika   MeSH
• fylogeneze MeSH
• proteiny červů chemie   genetika   MeSH
• sekvence aminokyselin MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cystatiny * MeSH
• disulfidy MeSH
• proteiny červů MeSH

Tick saliva is a rich source of antihemostatic, anti-inflammatory, and immunomodulatory molecules that actively help the tick to finish its blood meal. Moreover, these molecules facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-8, a salivary serpin from the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Iripin-8 displayed blood-meal-induced mRNA expression that peaked in nymphs and the salivary glands of adult females. Iripin-8 inhibited multiple proteases involved in blood coagulation and blocked the intrinsic and common pathways of the coagulation cascade in vitro. Moreover, Iripin-8 inhibited erythrocyte lysis by complement, and Iripin-8 knockdown by RNA interference in tick nymphs delayed the feeding time. Finally, we resolved the crystal structure of Iripin-8 at 1.89 Å resolution to reveal an unusually long and rigid reactive center loop that is conserved in several tick species. The P1 Arg residue is held in place distant from the serpin body by a conserved poly-Pro element on the P' side. Several PEG molecules bind to Iripin-8, including one in a deep cavity, perhaps indicating the presence of a small-molecule binding site. This is the first crystal structure of a tick serpin in the native state, and Iripin-8 is a tick serpin with a conserved reactive center loop that possesses antihemostatic activity that may mediate interference with host innate immunity.

• Klíčová slova
• Ixodes ricinus, blood coagulation, crystal structure, parasite, saliva, serpin, tick,
• MeSH
• aktivace komplementu účinky léků   imunologie   fyziologie   MeSH
• erytrocyty metabolismus   MeSH
• exprese genu genetika   MeSH
• hemokoagulace účinky léků   fyziologie   MeSH
• klíště enzymologie   genetika   metabolismus   MeSH
• komplement metabolismus   MeSH
• lymeská nemoc MeSH
• nymfa MeSH
• proteiny členovců metabolismus   MeSH
• regulace genové exprese genetika   MeSH
• serpiny metabolismus   ultrastruktura   MeSH
• slinné žlázy metabolismus   MeSH
• sliny chemie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• komplement MeSH
• proteiny členovců MeSH
• serpiny 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

Tick saliva is a rich source of pharmacologically and immunologically active molecules. These salivary components are indispensable for successful blood feeding on vertebrate hosts and are believed to facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-3, a protein expressed in the salivary glands of the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Belonging to the serpin superfamily of protease inhibitors, Iripin-3 strongly inhibited the proteolytic activity of serine proteases kallikrein and matriptase. In an in vitro setup, Iripin-3 was capable of modulating the adaptive immune response as evidenced by reduced survival of mouse splenocytes, impaired proliferation of CD4+ T lymphocytes, suppression of the T helper type 1 immune response, and induction of regulatory T cell differentiation. Apart from altering acquired immunity, Iripin-3 also inhibited the extrinsic blood coagulation pathway and reduced the production of pro-inflammatory cytokine interleukin-6 by lipopolysaccharide-stimulated bone marrow-derived macrophages. In addition to its functional characterization, we present the crystal structure of cleaved Iripin-3 at 1.95 Å resolution. Iripin-3 proved to be a pluripotent salivary serpin with immunomodulatory and anti-hemostatic properties that could facilitate tick feeding via the suppression of host anti-tick defenses. Physiological relevance of Iripin-3 activities observed in vitro needs to be supported by appropriate in vivo experiments.

• Klíčová slova
• Ixodes ricinus, X-ray crystallography, adaptive immunity, blood coagulation, inflammation, saliva, serpin, tick,
• MeSH
• adaptivní imunita účinky léků   MeSH
• aktivace lymfocytů účinky léků   MeSH
• antikoagulancia izolace a purifikace   farmakologie   MeSH
• cytokiny metabolismus   MeSH
• hemokoagulace účinky léků   MeSH
• hmyzí proteiny izolace a purifikace   farmakologie   MeSH
• imunologické faktory izolace a purifikace   farmakologie   MeSH
• inhibitory proteas izolace a purifikace   farmakologie   MeSH
• klíště metabolismus   MeSH
• králíci MeSH
• kultivované buňky MeSH
• lidé MeSH
• lymfocyty účinky léků   imunologie   metabolismus   MeSH
• morčata MeSH
• myši inbrední C3H MeSH
• myši inbrední C57BL MeSH
• myši transgenní MeSH
• myši MeSH
• proliferace buněk účinky léků   MeSH
• slezina účinky léků   imunologie   metabolismus   MeSH
• slinné proteiny a peptidy izolace a purifikace   farmakologie   MeSH
• sliny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• lidé MeSH
• morčata MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antikoagulancia MeSH
• cytokiny MeSH
• hmyzí proteiny MeSH
• imunologické faktory MeSH
• inhibitory proteas MeSH
• slinné proteiny a peptidy MeSH