Amblyostatin-1, the first salivary cystatin with host immunomodulatory and anti-inflammatory properties from the Neotropical tick Amblyomma sculptum, vector of Brazilian spotted fever

Wilson Santos Molari¹Mohamed Amine Jmel²Josiane Betim de Assis¹Alan Frazão Silva¹Júlia de Moura Bernardi¹Gretta Huamanrayme Bustamante¹José María Medina³Eliane Esteves⁴Solange Antão Antão⁵Andréa Cristina Fogaça⁵Zdenek Franta⁶Lucas Tirloni⁷Michail Kotsyfakis^2,8Anderson Sa-Nunes^1*

• ¹Department of Immunology, University of São Paulo, São Paulo, Brazil
• ²Institute of Parasitology, Centre for Biology, Academy of Sciences of the Czech Republic (ASCR), České Budějovice, South Bohemia, Czechia
• ³s/n, 18071 Granada, Spain, Facultad de Ciencias, Universidad de Granada, Campus de Fuentenueva, s/n, 18071 Granada, Spain
• ⁴Department of Microbiology and Immunology, Frederick P. Whiddon College of Medicine, University of South Alabama, Mobile AL, United States
• ⁵Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, São Paulo, Brazil
• ⁶Department of Botany, Faculty of Science, University of South Bohemia, Ceske Budejovice, South Bohemia, Czechia
• ⁷Tick-Pathogen Transmission Unit, Laboratory of Bacteriology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, Hamilton, MT, United States
• ⁸Institute of Molecular Biology and Biotechnology (IMBB), Foundation for Research and Technology Hellas (FORTH), Crete, Greece

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. 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 uM) 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. These findings establish Amblyostatin-1 as a novel salivary cystatin with immunomodulatory and anti-inflammatory properties, presenting potential as an immunobiological agent.