Functional study of protease BmSEI present in the gut of the tick Rhipicephalus microplus and their participation in infection with Anaplasma marginale.

• Grant number: 13/01175-9
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: May 01, 2013
• End date: April 30, 2016
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Aparecida Sadae Tanaka
• Grantee: Cícera Maria Gomes
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated research grant: 12/03657-8 - Inhibitor and proteases of ectoparasites: relationship of structure-function and identification of the role of these molecules in the interaction of diseases vector e their etiological agents, AP.TEM

Abstract

Ticks are important vectors of diseases to humans and other vertebrates. The Rhipicephalus (Boophilus) microplus is an exclusive ectoparasite of cattle, being responsible for transmission of Anaplasma sp and Babesia sp, etiologic agents of the disease called "tick fever". The R. microplus causes great damage to livestock, resulting in considerable losses in the production of meat, milk and leather. The ectoparasite control using acaricides is the main method, however it can lead to bovine contamination, as well as the environment. An alternative method to tick control is the production of vaccine, however, to date no vaccine appeared effective for Brazilian cattle. In attempt to identify important proteins for R. microplus, which may compose a multi-vaccine antigens, the aims of this work are: to study an inhibitor of human neutrophil elastase (BmSEI). This inhibitor was first identified in the saliva of ticks, and later the transcript encoding this protein was obtained from transcriptome analysis of intestine of R. microplus engorged females. In order to elucidate the function of this protein, a cDNA library from gut was constructed and about 800 clones were sequenced. After a preliminary analysis, we observed a high frequency of transcripts encoding proteins similar to BmSEI (Boophilus microplus saliva elastase inhibitor, GenBank accession number: ABH10604.1), suggesting a possible role of these proteins in this organ. In this work, a recombinant protein BmSEI will be produced and characterized regarding inhibitory activity of proteases and possible antimicrobial function. BmSEI expression analysis in different tissues will be conducted through quantitative real time PCR (qRT-PCR). In order to determine antimicrobial role, we are going to silence BmSEI transcript by RNA interference followed by qPCR. Symbiont bacteria from the gut and eggs of BmSEI silenced ticks will be quantified by qPCR in comparison with control group. Moreover, we are going to evaluate production of eggs of ticks silencing and also silenced and followed by E. coli or M. luteus infection. Finally, BmSEI silencing in R. microplus BME26 cell culture and comparison of the molecular repertoire of cells silenced and non silenced followed by infection with A. marginale will be performed by RNA sequencing.