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.

Departamento de Genética Facultad de Ciencias Universidad de Granada Granada Spain

Department of Biochemistry Federal University of São Paulo São Paulo Brazil

Department of Chemistry Faculty of Science University of South Bohemia České Budějovice Czechia

Department of Immunology Institute of Biomedical Sciences University of São Paulo São Paulo Brazil

Department of Microbiology and Immunology Frederick P Whiddon College of Medicine University of South Alabama Mobile AL United States

Department of Parasitology Institute of Biomedical Sciences University of São Paulo São Paulo SP Brazil

Institute of Molecular Biology and Biotechnology Foundation for Research and Technology Hellas Heraklion Greece

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice Czechia

National Institute of Science and Technology in Molecular Entomology National Council for Scientific and Technological Development Rio de Janeiro Brazil

Tick Pathogen Transmission Unit Laboratory of Bacteriology Division of Intramural Research National Institute of Allergy and Infectious Diseases Hamilton MT United States

Université de Lorraine CNRS IMoPA Nancy France

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