Ixodes scapularis is a primary vector of several important tick-borne pathogens including Borrelia burgdorferi sensu lato, the causative bacterial genospecies complex of Lyme disease, Babesia microti, Anaplasma phagocytophilum, Borrelia miyamotoi, Ehrlichia muris eauclarensis, and Powassan virus. Salivary compounds secreted by I. scapularis during blood feeding are immunogenic and can elicit robust antibody responses in humans which can potentially be leveraged as surrogate markers of prior tick bite exposure. In this study, we investigate the potential of a tick secreted salivary serine protease inhibitor, IxsS7, as a novel antigenic biomarker of I. scapularis exposure in humans. We demonstrate that the IxsS7 protein-coding sequence is highly conserved (>90 % identity) among other important Ixodes species (e.g., Ixodes ricinus, Ixodes persulcatus, and Ixodes pacificus) and poorly conserved (<50 % identity) with homologs from other tick genera, such as Amblyomma spp., Dermacentor spp., Rhipicephalus spp., and Haemaphysalis spp. Antibodies in sera from rabbits immunized with recombinant IxsS7 (rIxsS7) strongly recognize native IxsS7 when challenged with salivary gland homogenate (SGH) from blood-fed I. scapularis females, while showing minimal cross-reactivity with SGH from other hard tick (Ixodidae) genera. Western blot and ELISA analyses revealed that human subjects who reported recent prior exposure to ticks possessed IgG antibodies that recognized rIxsS7, highlighting its potential as a biomarker of exposure specifically against I. scapularis. Further development of serological tools that can measure human antibody responses to Ixodes-specific salivary antigens is essential to better quantify individual- and population-level risk of important tick-borne diseases such as Lyme disease.

• Keywords
• Borrelia, Ixodes, Ixodid, Lyme, Tick, Tick saliva, Vaccine,
• MeSH
• Biomarkers blood   MeSH
• Ixodes * genetics   MeSH
• Tick Bites * diagnosis   MeSH
• Humans MeSH
• Arthropod Proteins * genetics   immunology   MeSH
• Salivary Proteins and Peptides * genetics   immunology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Biomarkers MeSH
• Arthropod Proteins * MeSH
• Salivary Proteins and Peptides * MeSH

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.

• Keywords
• Comparative genomics, Duplication, Hematophagy, Parasite, Retroposition,
• MeSH
• Biological Evolution * MeSH
• Phylogeny MeSH
• Genome * MeSH
• Ixodes * genetics   classification   MeSH
• Evolution, Molecular * MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article 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.

• Keywords
• autoimmune disease, immune responses, protease inhibitors, psoriasis, tick,
• MeSH
• Dermatitis * MeSH
• Endopeptidases MeSH
• Cysteine Proteinase Inhibitors MeSH
• Protease Inhibitors MeSH
• Humans MeSH
• Mannans MeSH
• Immunity, Innate MeSH
• Peptide Hydrolases MeSH
• Psoriasis * chemically induced   drug therapy   MeSH
• Inflammation drug therapy   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Endopeptidases MeSH
• Cysteine Proteinase Inhibitors MeSH
• Protease Inhibitors MeSH
• Mannans MeSH
• Peptide Hydrolases 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.

• Keywords
• Cystatins, Host–parasite interactions, Ixodes ricinus, Protease inhibition, Protein structure, Tick saliva,
• MeSH
• Cystatins * pharmacology   MeSH
• Cysteine metabolism   MeSH
• Endopeptidases metabolism   MeSH
• Cathepsins metabolism   MeSH
• Ixodes * chemistry   MeSH
• Vertebrates MeSH
• Peptide Hydrolases metabolism   MeSH
• Salivary Cystatins chemistry   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cystatins * MeSH
• Cysteine MeSH
• Endopeptidases MeSH
• Cathepsins MeSH
• Peptide Hydrolases MeSH
• Salivary Cystatins MeSH

Iripin-4, one of the many salivary serpins from Ixodes ricinus ticks with an as-yet unexplained function, crystallized in two different structural conformations, namely the native partially relaxed state and the cleaved serpin. The native structure was solved at a resolution of 2.3 Å and the structure of the cleaved conformation was solved at 2.0 Å resolution. Furthermore, structural changes were observed when the reactive-centre loop transitioned from the native conformation to the cleaved conformation. In addition to this finding, it was confirmed that Glu341 represents a primary substrate-recognition site for the inhibitory mechanism. The presence of glutamate instead of the typical arginine in the P1 recognition site of all structurally characterized I. ricinus serpins (PDB entries 7b2t, 7pmu and 7ahp), except for the tyrosine in the P1 site of Iripin-2 (formerly IRS-2; PDB entry 3nda), would explain the absence of inhibition of the tested proteases that cleave their substrate after arginine. Further research on Iripin-4 should focus on functional analysis of this interesting serpin.

• Keywords
• Iripin-4, Ixodes ricinus, X-ray structure, cleaved conformation, native conformation, serpins,
• MeSH
• Arginine MeSH
• Ixodes * MeSH
• Protein Conformation MeSH
• Models, Molecular MeSH
• Serpins * chemistry   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Arginine MeSH
• Serpins * MeSH

Serpins are widely distributed and functionally diverse inhibitors of serine proteases. Ticks secrete serpins with anti-coagulation, anti-inflammatory, and immunomodulatory activities via their saliva into the feeding cavity to modulate host's hemostatic and immune reaction initiated by the insertion of tick's mouthparts into skin. The suppression of the host's immune response not only allows ticks to feed on a host for several days but also creates favorable conditions for the transmission of tick-borne pathogens. Herein we present the functional and structural characterization of Iripin-1 (Ixodes ricinus serpin-1), whose expression was detected in the salivary glands of the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Of 16 selected serine proteases, Iripin-1 inhibited primarily trypsin and further exhibited weaker inhibitory activity against kallikrein, matriptase, and plasmin. In the mouse model of acute peritonitis, Iripin-1 enhanced the production of the anti-inflammatory cytokine IL-10 and chemokines involved in neutrophil and monocyte recruitment, including MCP-1/CCL2, a potent histamine-releasing factor. Despite increased chemokine levels, the migration of neutrophils and monocytes to inflamed peritoneal cavities was significantly attenuated following Iripin-1 administration. Based on the results of in vitro experiments, immune cell recruitment might be inhibited due to Iripin-1-mediated reduction of the expression of chemokine receptors in neutrophils and adhesion molecules in endothelial cells. Decreased activity of serine proteases in the presence of Iripin-1 could further impede cell migration to the site of inflammation. Finally, we determined the tertiary structure of native Iripin-1 at 2.10 Å resolution by employing the X-ray crystallography technique. In conclusion, our data indicate that Iripin-1 facilitates I. ricinus feeding by attenuating the host's inflammatory response at the tick attachment site.

• Keywords
• anti-inflammatory protein, cell migration, iripin, ixodes ricinus, serpin, tick saliva, tick-host interaction, ticks,
• MeSH
• Anti-Inflammatory Agents pharmacology   MeSH
• Chemokines MeSH
• Endothelial Cells metabolism   MeSH
• Ixodes * metabolism   MeSH
• Monocytes metabolism   MeSH
• Mice MeSH
• Serpins * metabolism   MeSH
• Trypsin MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• Anti-Inflammatory Agents MeSH
• Chemokines MeSH
• Serpins * MeSH
• Trypsin MeSH

Tick saliva has been extensively studied in the context of tick-host interactions because it is involved in host homeostasis modulation and microbial pathogen transmission to the host. Accumulated knowledge about the tick saliva composition at the molecular level has revealed that serine protease inhibitors play a key role in the tick-host interaction. Serpins are one highly expressed group of protease inhibitors in tick salivary glands, their expression can be induced during tick blood-feeding, and they have many biological functions at the tick-host interface. Indeed, tick serpins have an important role in inhibiting host hemostatic processes and in the modulation of the innate and adaptive immune responses of their vertebrate hosts. Tick serpins have also been studied as potential candidates for therapeutic use and vaccine development. In this review, we critically summarize the current state of knowledge about the biological role of tick serpins in shaping tick-host interactions with emphasis on the mechanisms by which they modulate host immunity. Their potential use in drug and vaccine development is also discussed.

• Keywords
• anti-tick vaccine, immunomodulation, serpins, therapeutic effects, tick host interaction, tick saliva,
• MeSH
• Serine Proteinase Inhibitors physiology   MeSH
• Ticks * metabolism   MeSH
• Serpins * metabolism   MeSH
• Salivary Glands metabolism   MeSH
• Saliva metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Serine Proteinase Inhibitors MeSH
• Serpins * MeSH

Hemostatic disorders are caused either by platelet-related dysfunctions, defective blood coagulation, or by a combination of both, leading to an increased susceptibility to cardiovascular diseases (CVD) and other related illnesses. The unique specificity of anticoagulants from hematophagous arthropods, such as ticks, suggests that tick saliva holds great promise for discovering new treatments for these life-threatening diseases. In this study, we combined in silico and in vitro analyses to characterize the first recombinant serpin, herein called Dromaserpin, from the sialotranscriptome of the Hyalomma dromedarii tick. Our in silico data described Dromaserpin as a secreted protein of ~43 kDa with high similarities to previously characterized inhibitory serpins. The recombinant protein (rDromaserpin) was obtained as a well-structured monomer, which was tested using global blood coagulation and platelet aggregation assays. With this approach, we confirmed rDromaserpin anticoagulant activity as it significantly delayed plasma clotting in activated partial thromboplastin time and thrombin time assays. The profiling of proteolytic activity shows its capacity to inhibit thrombin in the micromolar range (0.2 to 1 μM) and in the presence of heparin this inhibition was clearly increased. It was also able to inhibit Kallikrein, FXIa and slightly FXIIa, with no significant effect on other factors. In addition, the rDromaserpin inhibited thrombin-induced platelet aggregation. Taken together, our data suggest that rDromaserpin deserves to be further investigated as a potential candidate for developing therapeutic compounds targeting disorders related to blood clotting and/or platelet aggregation.

• Keywords
• Hyalomma dromedarii, anticoagulants, salivary glands, serpin, thrombin inhibitor,
• MeSH
• Anticoagulants chemistry   metabolism   MeSH
• Phylogeny MeSH
• Blood Coagulation drug effects   MeSH
• Ixodidae metabolism   MeSH
• Protein Conformation MeSH
• Models, Molecular MeSH
• Computer Simulation MeSH
• Amino Acid Sequence MeSH
• Serpins chemistry   metabolism   pharmacology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anticoagulants MeSH
• Serpins MeSH

Iripin-5 is the main Ixodes ricinus salivary serpin, which acts as a modulator of host defence mechanisms by impairing neutrophil migration, suppressing nitric oxide production by macrophages and altering complement functions. Iripin-5 influences host immunity and shows high expression in the salivary glands. Here, the crystal structure of Iripin-5 in the most thermodynamically stable state of serpins is described. In the reactive-centre loop, the main substrate-recognition site of Iripin-5 is likely to be represented by Arg342, which implies the targeting of trypsin-like proteases. Furthermore, a computational structural analysis of selected Iripin-5-protease complexes together with interface analysis revealed the most probable residues of Iripin-5 involved in complex formation.

• Keywords
• Iripin-5, Ixodes ricinus, X-ray structure, serine protease inhibitors, serpins, tick saliva,
• MeSH
• Anti-Inflammatory Agents * chemistry   isolation & purification   MeSH
• Erythrocytes MeSH
• Enzyme Inhibitors * chemistry   isolation & purification   MeSH
• Ixodes metabolism   MeSH
• Rabbits MeSH
• Cells, Cultured MeSH
• Macrophages MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Neutrophils MeSH
• Serpins * chemistry   isolation & purification   MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anti-Inflammatory Agents * MeSH
• Enzyme Inhibitors * MeSH
• Serpins * 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.

• Keywords
• Ixodes ricinus, blood coagulation, crystal structure, parasite, saliva, serpin, tick,
• MeSH
• Complement Activation drug effects   immunology   physiology   MeSH
• Erythrocytes metabolism   MeSH
• Gene Expression genetics   MeSH
• Blood Coagulation drug effects   physiology   MeSH
• Ixodes enzymology   genetics   metabolism   MeSH
• Complement System Proteins metabolism   MeSH
• Lyme Disease MeSH
• Nymph MeSH
• Arthropod Proteins metabolism   MeSH
• Gene Expression Regulation genetics   MeSH
• Serpins metabolism   ultrastructure   MeSH
• Salivary Glands metabolism   MeSH
• Saliva chemistry   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Complement System Proteins MeSH
• Arthropod Proteins MeSH
• Serpins MeSH