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.

• Klíčová slova
• Amblyomma sculptum, Amblyostatin-1, immunomodulation, inflammation, tick saliva, tick-host interaction,
• MeSH
• Amblyomma * imunologie   metabolismus   MeSH
• antiflogistika * farmakologie   MeSH
• arachnida jako vektory * imunologie   MeSH
• cystatiny * imunologie   MeSH
• dendritické buňky imunologie   účinky léků   MeSH
• myši MeSH
• proteiny členovců * genetika   imunologie   MeSH
• slinné cystatiny * genetika   imunologie   farmakologie   chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antiflogistika * MeSH
• cystatiny * MeSH
• proteiny členovců * MeSH
• slinné cystatiny * 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

Protease inhibitors (PIs) are ubiquitous regulatory proteins present in all kingdoms. They play crucial tasks in controlling biological processes directed by proteases which, if not tightly regulated, can damage the host organism. PIs can be classified according to their targeted proteases or their mechanism of action. The functions of many PIs have now been characterized and are showing clinical relevance for the treatment of human diseases such as arthritis, hepatitis, cancer, AIDS, and cardiovascular diseases, amongst others. Other PIs have potential use in agriculture as insecticides, anti-fungal, and antibacterial agents. PIs from tick salivary glands are special due to their pharmacological properties and their high specificity, selectivity, and affinity to their target proteases at the tick-host interface. In this review, we discuss the structure and function of PIs in general and those PI superfamilies abundant in tick salivary glands to illustrate their possible practical applications. In doing so, we describe tick salivary PIs that are showing promise as drug candidates, highlighting the most promising ones tested in vivo and which are now progressing to preclinical and clinical trials.

• Klíčová slova
• drug discovery, protease inhibitors, proteases, tick saliva,
• MeSH
• inhibitory proteas izolace a purifikace   terapeutické užití   MeSH
• interakce hostitele a parazita genetika   imunologie   MeSH
• klíšťata metabolismus   MeSH
• lidé MeSH
• slinné žlázy metabolismus   MeSH
• sliny chemie   metabolismus   MeSH
• transkriptom genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• inhibitory proteas MeSH