Functional characterization of a immunogenic salivary Trypsin-inhibitor-like protein from the "Cattle tick" Rhipicephalus microplus

• Grant number: 23/12568-3
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Start date: February 05, 2024
• End date: May 04, 2024
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Aparecida Sadae Tanaka
• Grantee: Gabriel Cerqueira Alves Costa
• Supervisor: Lucas Tirloni
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Institution abroad: Rocky Mountain Laboratories, United States
• Associated to the scholarship: 20/02433-5 - Library construction of cDNA fragments of R. microplus proteolytic enzymes (peptidases) and serpins on the surface of filamentous phages, BP.PD

Abstract

Rhipicephalus microplus, also known as cattle tick, is one of the most important tick species in Brazil. This ectoparasite is related to severe infestations which can cause significative harm to livestock animals, resulting in expressive economic losses in tropical and subtropical countries. The main tick control strategy consists in treating animals with chemical acaricides, which present different drawbacks, including the selection of resistant tick populations and environmental pollution. Thus, alternative control methods have been studied and one of the most promisor approaches are anti-tick vaccines, which presented satisfactory results against R. microplus parasitism. Nevertheless, the discovery of new immunogenic tick molecules is required for the development of new efficient formulations for Brazilian populations. Then, the main goal of the present study is to functionally characterize an immunogenic protein present in saliva of female ticks during hematophagy. This candidate was identified using the high-throughput technics of phage display and Nanopore DNA sequencing technology. A salivary ORFeome phage display library was screened against anti-tick saliva IgG antibodies elicited in cattle experimentally and repeatedly infested with R. microplus. The most frequent peptide corresponded to a cysteine-rich protein containing a trypsin inhibitor-like (TIL) domain, named here as RmTIL. This protein will be functionally characterized according to its enzymatic inhibition against different serine proteases, including bovine enzymes expressed by Dr. Tirloni group in the Rocky Mountain Laboratories (RML). Moreover, inhibition of wound healing process, vascular permeability, and edema in vertebrate's skin will also be evaluated in collaboration with Dr. Tirloni. Finally, immunization assays will be executed to verify the recombinant protein immunogenicity and the elicited antibodies will be quantified. The functional analysis might contribute by adding new information about the importance of this salivary protein to tick-host interaction and its potential to be applied on the development of new control strategies against the R. microplus.