INTRODUCTION: The Neotropical tick Amblyomma sculptum is the primary vector of Rickettsia rickettsii, the causative agent of Brazilian spotted fever, a disease associated with high fatality rates. Tick saliva, a complex mixture of bioactive molecules essential for successful blood feeding, facilitates pathogen transmission and modulates host immune responses. A comprehensive evaluation of the salivary gland transcriptome database reveals that protease inhibitors are abundantly expressed molecules in tick saliva during feeding. Thus, this study aims to describe and characterize the most expressed member of the cystatin family identified in Amblyomma sculptum salivary transcriptome, named Amblyostatin-1. METHODS: Bioinformatic tools were employed for in silico analysis of the Amblyostatin-1 sequence and structure. A recombinant version of Amblyostatin-1 was expressed in an Escherichia coli system, evaluated against a panel of cysteine proteases in biochemical assays, and used to generate antibodies in immunized mice. The biological activities of Amblyostatin-1 were assessed by its effects on dendritic cell maturation in vitro and in a carrageenan-induced inflammation model in vivo. RESULTS: Based on its sequence and predicted three-dimensional structure, Amblyostatin-1 is classified as an I25B cystatin, and its recombinant form selectively inhibits cathepsins L, C, and S at different rates, with a low nanomolar Ki value of 0.697 ± 0.22 nM against cathepsin L. Regarding its biological activities, recombinant Amblyostatin-1 partially affects LPS-induced dendritic cell maturation by downmodulating the costimulatory molecules CD80 and CD86 at higher micromolar concentrations (3 µM) while promoting IL-10 production at nanomolar concentrations (100 nM). The apparent lack of Amblyostatin-1-specific antibody responses in immunized mice suggests an impairment of antigen processing and presentation in vivo. Furthermore, in a carrageenan-induced inflammation model, Amblyostatin-1 decreased edema formation and neutrophil infiltration into the skin without affecting other myeloid cells. DISCUSSION: These findings establish Amblyostatin-1 as a novel salivary cystatin with immunomodulatory and anti-inflammatory properties, highlighting its potential as an immunobiological agent.

• Keywords
• Amblyomma sculptum, Amblyostatin-1, immunomodulation, inflammation, tick saliva, tick-host interaction,
• MeSH
• Amblyomma * immunology   metabolism   MeSH
• Anti-Inflammatory Agents * pharmacology   MeSH
• Arachnid Vectors * immunology   MeSH
• Cystatins * immunology   MeSH
• Dendritic Cells immunology   drug effects   MeSH
• Mice MeSH
• Arthropod Proteins * genetics   immunology   MeSH
• Salivary Cystatins * genetics   immunology   pharmacology   chemistry   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anti-Inflammatory Agents * MeSH
• Cystatins * MeSH
• Arthropod Proteins * MeSH
• Salivary Cystatins * 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.

• Keywords
• Ixodes ricinus, cathepsin, crystal structure, cysteine protease, digestion, midgut, parasite,
• MeSH
• Cystatins metabolism   MeSH
• Phylogeny MeSH
• Cathepsin L metabolism   MeSH
• Ticks metabolism   MeSH
• Ixodes metabolism   MeSH
• Blood Proteins metabolism   MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Proteolysis MeSH
• Amino Acid Sequence MeSH
• Digestive System metabolism   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cystatins MeSH
• Cathepsin L MeSH
• Blood Proteins MeSH

Parasite inhibitors of cysteine peptidases are known to influence a vast range of processes linked to a degradation of either the parasites' own proteins or proteins native to their hosts. We characterise a novel type I cystatin (stefin) found in a sanguinivorous fish parasite Eudiplozoon nipponicum (Platyhelminthes: Monogenea). We have identified a transcript of its coding gene in the transcriptome of adult worms. Its amino acid sequence is similar to other stefins except for containing a legumain-binding domain, which is in this type of cystatins rather unusual. As expected, the recombinant form of E. nipponicum stefin (rEnStef) produced in Escherichia coli inhibits clan CA peptidases - cathepsins L and B of the worm - via the standard papain-binding domain. It also blocks haemoglobinolysis by cysteine peptidases in the worm's excretory-secretory products and soluble extracts. Furthermore, we had confirmed its ability to inhibit clan CD asparaginyl endopeptidase (legumain). The presence of a native EnStef in the excretory-secretory products of adult worms, detected by mass spectrometry, suggests that this protein has an important biological function at the host-parasite interface. We discuss the inhibitor's possible role in the regulation of blood digestion, modulation of antigen presentation, and in the regeneration of host tissues.

• MeSH
• Cystatins metabolism   MeSH
• Cysteine Endopeptidases metabolism   MeSH
• Escherichia coli MeSH
• Phylogeny MeSH
• Carps parasitology   MeSH
• Cloning, Molecular MeSH
• Protein Conformation MeSH
• Platyhelminths metabolism   MeSH
• Computer Simulation MeSH
• Protein Domains MeSH
• Helminth Proteins genetics   metabolism   MeSH
• Recombinant Proteins genetics   metabolism   MeSH
• Sequence Analysis, Protein MeSH
• Sequence Alignment MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• asparaginylendopeptidase MeSH Browser
• Cystatins MeSH
• Cysteine Endopeptidases MeSH
• Helminth Proteins MeSH
• Recombinant Proteins MeSH

To ensure successful feeding tick saliva contains a number of inhibitory proteins that interfere with the host immune response and help to create a permissive environment for pathogen transmission. Among the potential targets of the salivary cystatins are two host cysteine proteases, cathepsin S, which is essential for antigen- and invariant chain-processing, and cathepsin C (dipeptidyl peptidase 1, DPP1), which plays a critical role in processing and activation of the granule serine proteases. Here, the effect of salivary cystatin OmC2 from Ornithodoros moubata was studied using differentiated MUTZ-3 cells as a model of immature dendritic cells of the host skin. Following internalization, cystatin OmC2 was initially found to inhibit the activity of several cysteine cathepsins, as indicated by the decreased rates of degradation of fluorogenic peptide substrates. To identify targets, affinity chromatography was used to isolate His-tagged cystatin OmC2 together with the bound proteins from MUTZ-3 cells. Cathepsins S and C were identified in these complexes by mass spectrometry and confirmed by immunoblotting. Furthermore, reduced increase in the surface expression of MHC II and CD86, which are associated with the maturation of dendritic cells, was observed. In contrast, human inhibitor cystatin C, which is normally expressed and secreted by dendritic cells, did not affect the expression of CD86. It is proposed that internalization of salivary cystatin OmC2 by the host dendritic cells targets cathepsins S and C, thereby affecting their maturation.

• Keywords
• DPP1, cathepsin C, cathepsin S, cystatin OmC2, dendritic cells, dipeptidyl peptidase 1, lysosomal proteases, tick saliva,
• MeSH
• B7-2 Antigen MeSH
• Antigens, Differentiation, B-Lymphocyte MeSH
• Cell Line MeSH
• Cystatins metabolism   MeSH
• Dendritic Cells immunology   metabolism   MeSH
• Epoxy Compounds immunology   metabolism   MeSH
• Genes, MHC Class II immunology   MeSH
• Cathepsin C metabolism   MeSH
• Cathepsins chemistry   immunology   metabolism   MeSH
• Ticks enzymology   MeSH
• Humans MeSH
• Lysosomes enzymology   MeSH
• Histocompatibility Antigens Class II MeSH
• Ornithodoros enzymology   MeSH
• Recombinant Proteins MeSH
• Saliva enzymology   MeSH
• Tyrosine analogs & derivatives   immunology   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• B7-2 Antigen MeSH
• Antigens, Differentiation, B-Lymphocyte MeSH
• cathepsin S MeSH Browser
• cathestatin C MeSH Browser
• CTSC protein, human MeSH Browser
• Cystatins MeSH
• Epoxy Compounds MeSH
• invariant chain MeSH Browser
• Cathepsin C MeSH
• Cathepsins MeSH
• Histocompatibility Antigens Class II MeSH
• Recombinant Proteins MeSH
• Tyrosine MeSH

The publication of the first tick sialome (salivary gland transcriptome) heralded a new era of research of tick protease inhibitors, which represent important constituents of the proteins secreted via tick saliva into the host. Three major groups of protease inhibitors are secreted into saliva: Kunitz inhibitors, serpins, and cystatins. Kunitz inhibitors are anti-hemostatic agents and tens of proteins with one or more Kunitz domains are known to block host coagulation and/or platelet aggregation. Serpins and cystatins are also anti-hemostatic effectors, but intriguingly, from the translational perspective, also act as pluripotent modulators of the host immune system. Here we focus especially on this latter aspect of protease inhibition by ticks and describe the current knowledge and data on secreted salivary serpins and cystatins and their role in tick-host-pathogen interaction triad. We also discuss the potential therapeutic use of tick protease inhibitors.

• Keywords
• cystatins, immunomodulation, protease inhibitors, serpins, tick-host interaction,
• MeSH
• Cystatins physiology   therapeutic use   MeSH
• Immunomodulation MeSH
• Protease Inhibitors classification   metabolism   therapeutic use   MeSH
• Serine Proteinase Inhibitors physiology   therapeutic use   MeSH
• Host-Parasite Interactions MeSH
• Ticks metabolism   MeSH
• Humans MeSH
• Serpins physiology   therapeutic use   MeSH
• Saliva enzymology   metabolism   MeSH
• Transcriptome MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Cystatins MeSH
• Protease Inhibitors MeSH
• Serine Proteinase Inhibitors MeSH
• Serpins 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
• Ixodes genetics   metabolism   MeSH
• Sequence Analysis, RNA * MeSH
• Cattle MeSH
• Gene Expression Profiling * MeSH
• Intestines pathology   MeSH
• Intestinal Mucosa metabolism   MeSH
• Transcriptome physiology   MeSH
• Animals MeSH
• Check Tag
• Cattle MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH

Haem and iron homeostasis in most eukaryotic cells is based on a balanced flux between haem biosynthesis and haem oxygenase-mediated degradation. Unlike most eukaryotes, ticks possess an incomplete haem biosynthetic pathway and, together with other (non-haematophagous) mites, lack a gene encoding haem oxygenase. We demonstrated, by membrane feeding, that ticks do not acquire bioavailable iron from haemoglobin-derived haem. However, ticks require dietary haemoglobin as an exogenous source of haem since, feeding with haemoglobin-depleted serum led to aborted embryogenesis. Supplementation of serum with haemoglobin fully restored egg fertility. Surprisingly, haemoglobin could be completely substituted by serum proteins for the provision of amino-acids in vitellogenesis. Acquired haem is distributed by haemolymph carrier protein(s) and sequestered by vitellins in the developing oocytes. This work extends, substantially, current knowledge of haem auxotrophy in ticks and underscores the importance of haem and iron metabolism as rational targets for anti-tick interventions.

• Keywords
• biochemistry, haem auxotrophy, haem oxygenase, haematophagy, infectious disease, iron metabolism, microbiology, ticks,
• MeSH
• Fertility MeSH
• Heme metabolism   MeSH
• Ticks metabolism   physiology   MeSH
• Reproduction MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Heme 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
• Transcription, Genetic physiology   MeSH
• Genome physiology   MeSH
• Hemoglobins genetics   metabolism   MeSH
• Cathepsin D genetics   metabolism   MeSH
• Ixodes enzymology   genetics   MeSH
• Protein Processing, Post-Translational physiology   MeSH
• Arthropod Proteins genetics   metabolism   MeSH
• Recombinant Proteins genetics   metabolism   MeSH
• Intestines enzymology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Hemoglobins MeSH
• Cathepsin D MeSH
• Arthropod Proteins MeSH
• Recombinant Proteins MeSH

Ticks, as obligate hematophagous ectoparasites, impact greatly on animal and human health because they transmit various pathogens worldwide. Over the last decade, several cystatins from different hard and soft ticks were identified and biochemically analyzed for their role in the physiology and blood feeding lifestyle of ticks. All these cystatins are potent inhibitors of papain-like cysteine proteases, but not of legumain. Tick cystatins were either detected in the salivary glands and/or the midgut, key tick organs responsible for blood digestion and the expression of pharmacologically potent salivary proteins for blood feeding. For example, the transcription of two cystatins named HlSC-1 and Sialostatin L2 was highly upregulated in these tick tissues during feeding. Vaccinating hosts against Sialostatin L2 and Om-cystatin 2 as well as silencing of a cystatin gene from Amblyomma americanum significantly inhibited the feeding ability of ticks. Additionally, Om-cystatin 2 and Sialostatin L possessed strong host immunosuppressive properties by inhibiting dendritic cell maturation due to their interaction with cathepsin S. These two cystatins, together with Sialostatin L2 are the first tick cystatins with resolved three-dimensional structure. Sialostatin L, furthermore, showed preventive properties against autoimmune diseases. In the case of the cystatin Hlcyst-2, experimental evidence showed its role in tick innate immunity, since increased Hlcyst-2 transcript levels were detected in Babesia gibsoni-infected larval ticks and the protein inhibited Babesia growth. Other cystatins, such as Hlcyst-1 or Om-cystatin 2 are assumed to be involved in regulating blood digestion. Only for Bmcystatin was a role in tick embryogenesis suggested. Finally, all the biochemically analyzed tick cystatins are powerful protease inhibitors, and some may be novel antigens for developing anti-tick vaccines and drugs of medical importance due to their stringent target specificity.

• MeSH
• Cystatins pharmacology   MeSH
• Cysteine Proteinase Inhibitors pharmacology   MeSH
• Ticks drug effects   physiology   MeSH
• Humans MeSH
• Models, Molecular MeSH
• Molecular Sequence Data MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Feeding Behavior drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Cystatins MeSH
• Cysteine Proteinase Inhibitors MeSH

Cysteine proteases have been discovered in various bloodfeeding ectoparasites. Here, we assemble the available information about the function of these peptidases and reveal their role in hematophagy and parasite development. While most of the data shed light on key proteolytic events that play a role in arthropod physiology, we also report on the association of cysteine proteases with arthropod vectorial capacity. With emphasis on ticks, specifically Ixodes ricinus, we finally propose a model about the contribution of cysteine peptidases to blood digestion and how their concerted action with other tick midgut proteases leads to the absorbance of nutrients by the midgut epithelial cells.

• MeSH
• Arthropods enzymology   MeSH
• Culicidae enzymology   MeSH
• Cysteine Proteases metabolism   MeSH
• Ticks enzymology   MeSH
• Parasites enzymology   MeSH
• Feeding Behavior physiology   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
• Cysteine Proteases MeSH