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

BACKGROUND: Alpha-Gal syndrome (AGS) is a tick-borne food allergy caused by IgE antibodies against the glycan galactose-alpha-1,3-galactose (α-Gal) present in glycoproteins and glycolipids from mammalian meat. To advance in the diagnosis and treatment of AGS, further research is needed to unravel the molecular and immune mechanisms underlying this syndrome. The objective of this study is the characterization of tick salivary components and proteins with and without α-Gal modifications involved in modulating human immune response against this carbohydrate. METHODS: Protein and α-Gal content were determined in tick saliva components, and proteins were identified by proteomics analysis of tick saliva fractions. Pathophysiological changes were recorded in the zebrafish (Danio rerio) model after exposure to distinct Ixodes ricinus tick salivary components. Serum samples were collected from zebrafish at day 8 of exposure to determine anti-α-Gal, anti-glycan, and anti-tick saliva protein IgM antibody titers by enzyme-linked immunosorbent assay (ELISA). RESULTS: Zebrafish treated with tick saliva and saliva protein fractions combined with non-protein fractions demonstrated significantly higher incidence of hemorrhagic type allergic reactions, abnormal behavioral patterns, or mortality when compared to the phosphate-buffered saline (PBS)-treated control group. The main tick salivary proteins identified in these fractions with possible functional implication in AGS were the secreted protein B7P208-salivary antigen p23 and metalloproteases. Anti-α-Gal and anti-tick salivary gland IgM antibody titers were significantly higher in distinct saliva protein fractions and deglycosylated saliva group when compared with PBS-treated controls. Anti-glycan antibodies showed group-related profiles. CONCLUSIONS: Results support the hypothesis that tick salivary biomolecules with and without α-Gal modifications are involved in modulating immune response against this carbohydrate.

• Keywords
• Allergy, Alpha-gal syndrome, Glycan, Tick, Zebrafish,
• MeSH
• Zebrafish metabolism   MeSH
• Galactose MeSH
• Immunoglobulin E MeSH
• Immunoglobulin M MeSH
• Ixodes * MeSH
• Tick Bites * MeSH
• Humans MeSH
• Food Hypersensitivity * etiology   MeSH
• Arthropod Proteins MeSH
• Mammals MeSH
• Saliva MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Galactose MeSH
• Immunoglobulin E MeSH
• Immunoglobulin M MeSH
• Arthropod Proteins 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

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.

• Keywords
• Ixodes ricinus, X-ray crystallography, adaptive immunity, blood coagulation, inflammation, saliva, serpin, tick,
• MeSH
• Adaptive Immunity drug effects   MeSH
• Lymphocyte Activation drug effects   MeSH
• Anticoagulants isolation & purification   pharmacology   MeSH
• Cytokines metabolism   MeSH
• Blood Coagulation drug effects   MeSH
• Insect Proteins isolation & purification   pharmacology   MeSH
• Immunologic Factors isolation & purification   pharmacology   MeSH
• Protease Inhibitors isolation & purification   pharmacology   MeSH
• Ixodes metabolism   MeSH
• Rabbits MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Lymphocytes drug effects   immunology   metabolism   MeSH
• Guinea Pigs MeSH
• Mice, Inbred C3H MeSH
• Mice, Inbred C57BL MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Cell Proliferation drug effects   MeSH
• Spleen drug effects   immunology   metabolism   MeSH
• Salivary Proteins and Peptides isolation & purification   pharmacology   MeSH
• Saliva metabolism   MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Humans MeSH
• Guinea Pigs MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anticoagulants MeSH
• Cytokines MeSH
• Insect Proteins MeSH
• Immunologic Factors MeSH
• Protease Inhibitors MeSH
• Salivary Proteins and Peptides MeSH

To successfully feed, ticks inject pharmacoactive molecules into the vertebrate host including cystatin cysteine protease inhibitors. However, the molecular and cellular events modulated by tick saliva remain largely unknown. Here, we describe and characterize a novel immunomodulatory cystatin, Iristatin, which is upregulated in the salivary glands of feeding Ixodes ricinus ticks. We present the crystal structure of Iristatin at 1.76 Å resolution. Purified recombinant Iristatin inhibited the proteolytic activity of cathepsins L and C and diminished IL-2, IL-4, IL-9, and IFN-γ production by different T-cell populations, IL-6 and IL-9 production by mast cells, and nitric oxide production by macrophages. Furthermore, Iristatin inhibited OVA antigen-induced CD4+ T-cell proliferation and leukocyte recruitment in vivo and in vitro. Our results indicate that Iristatin affects wide range of anti-tick immune responses in the vertebrate host and may be exploitable as an immunotherapeutic.

• Keywords
• Cathepsin, Crystal structure, Immune responses, Ixodes ricinus, Saliva,
• MeSH
• Cystatins classification   genetics   pharmacology   MeSH
• Cytokines metabolism   MeSH
• Epoxy Compounds metabolism   MeSH
• Phylogeny MeSH
• Immunosuppressive Agents chemistry   metabolism   pharmacology   MeSH
• Ixodes chemistry   genetics   metabolism   MeSH
• Crystallography, X-Ray MeSH
• Macrophages drug effects   metabolism   MeSH
• Nitric Oxide metabolism   MeSH
• Arthropod Proteins chemistry   genetics   pharmacology   MeSH
• Proteolysis drug effects   MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Salivary Cystatins chemistry   genetics   pharmacology   MeSH
• T-Lymphocytes drug effects   metabolism   MeSH
• Tyrosine analogs & derivatives   metabolism   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• cathestatin C MeSH Browser
• Cystatins MeSH
• Cytokines MeSH
• Epoxy Compounds MeSH
• Immunosuppressive Agents MeSH
• Nitric Oxide MeSH
• Arthropod Proteins MeSH
• Salivary Cystatins MeSH
• Tyrosine MeSH

UNLABELLED: Next generation sequencing and proteomics have helped to comprehensively characterize gene expression in tick salivary glands at both the transcriptome and the proteome level. Functional data are, however, lacking. Given that tick salivary secretions are critical to the success of the tick transmission lifecycle and, as a consequence, for host colonization by the pathogens they spread, we thoroughly review here the literature on the known interactions between tick saliva (or tick salivary gland extracts) and the innate and adaptive vertebrate immune system. The information is intended to serve as a reference for functional characterization of the numerous genes and proteins expressed in tick salivary glands with an ultimate goal to develop novel vector and pathogen control strategies. SIGNIFICANCE: We overview all the known interactions of tick saliva with the vertebrate immune system. The provided information is important, given the recent developments in high-throughput transcriptomic and proteomic analysis of gene expression in tick salivary glands, since it may serve as a guideline for the functional characterization of the numerous newly-discovered genes expressed in tick salivary glands.

• Keywords
• Adaptive immunity, Innate immunity, Saliva, Salivary glands, Tick,
• MeSH
• Insect Proteins immunology   MeSH
• Host-Parasite Interactions immunology   MeSH
• Ticks immunology   MeSH
• Models, Immunological MeSH
• Immunity, Innate immunology   MeSH
• Saliva immunology   metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• Insect Proteins MeSH