RmTIL, a cysteine-rich protein as a putative microbiota modulator from Rhipicephalus microplus tick

Rhipicephalus microplus , also known as the “cattle tick”, presents worldwide distribution in tropical and subtropical regions and causes significant economic losses in livestock activity. At the present work, an immunogenic cysteine-rich protein produced in salivary glands was identified through an association of phage display and third generation sequencing (TGS) high-throughput techniques. This protein presenting a trypsin inhibitor-like (TIL) domain from R. microplus , named here as RmTIL, was expressed as a recombinant protein and submitted to a functional characterization as a serine protease inhibitor. The recombinant protein corresponding to its TIL domain portion (rRmTIL-D) inhibited human neutrophil elastase, cathepsin G, and subtilisin A, displaying inhibition constant ( K i ) values of 7.0, 32.7 and 182.5 nM, respectively. RNA interference (RNAi) was employed to evaluate the effect of RmTIL-gene expression over reproductive parameters in engorged female ticks, revealing that eggs viability was significantly affected after gene silencing. Moreover, RmTIL gene silencing led to upregulation of 16S bacterial rRNA levels in ovary and eggs, while experimental infection of ticks with Gram-positive bacteria resulted in upregulated RmTIL transcripts. Following the increased bacterial burden, genes encoding proteins related to embryo development, lipid, redox, and energy metabolism pathways were modulated in eggs laid by RNAi-silenced females. Data presented here indicate that RmTIL is an immunogenic protein playing the two important roles of inhibiting host proteases and regulating tick microbiota, both crucial to the R. microplus life cycle.