Rmcystatin3, a cysteine protease inhibitor from Rhipicephalus microplus hemocytes involved in immune response

Abstract

The Rhipicephalus microplus tick is responsible for losses in the livestock production estimated in 2 billions USD. Despite its economical importance the knowledge in tick's physiology is sparse. In order to contribute to this scenario we describe the characterization of a cysteine proteinase inhibitor named Rmcystatin-3. Purified recombinant Rmcystatin-3 was able to inhibit cathepsin L (Ki = 2.5 nM), BmCl1 (Ki = 1.8 nM) and cathepsin B (Ki = 136 nM). Western blot and quantitative PCR analysis revealed the presence of Rmcystatin-3 in fat body, salivary gland but mainly in hemocytes. The mRNA levels of Rmcystatin-3 during bacterial challenge are drastically down-regulated. In order to define the Rmcystatin-3 possible role in tick immunity, the cystatin gene was knockdown by RNA interference with and without Escherichia coli infection. Our results showed that the Rmcystatin-3 silenced group was more immune competent to control bacterial infection than the group injected with non-related dsRNA. Taking together, our data strongly suggested an important role of Rmcystatin-3 in tick immunity.

Protease Inhibitors in Tick Saliva: The Role of Serpins and Cystatins in Tick-host-Pathogen Interaction

“…Rmcystatin3 inhibited cathepsins L and B and B oophilus m icroplus c athepsin L - 1 (BmCl1) (Lu et al, 2014). Bmcystatin3 transcripts were found in tick hemocytes, fat bodies, and salivary glands, but protein was only detected in hemocytes and the fat bodies by western blotting.…”

“…Bmcystatin3 transcripts were found in tick hemocytes, fat bodies, and salivary glands, but protein was only detected in hemocytes and the fat bodies by western blotting. Infection of ticks with E. coli significantly downregulated Bmcystatin3 expression (Lu et al, 2014) but increased efficacy of pathogen clearance, suggesting that Rmcystatin3 may be a negative regulator of tick immune responses, probably by regulating cysteine proteases responsible for the production of antimicrobial effectors in hemocytes (Lu et al, 2014). …”

A repertoire of protease inhibitor families in Amblyomma americanum and other tick species: inter-species comparative analyses

“…Studies characterizing the function of tick PIs show a role in host protease regulation [50, 60, 145, 146], and in the regulation of tick proteases [55, 83]. This study shows that there are at least 1,595 known putative non-redundant PIs in ixodid tick species.…”

RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control

“…The function of some components of immunity, such as α2-macroglobulin proteins [132,133], antimicrobial peptides [131], Janus kinase (JAK)-signaling transducer activator of transcription (STAT) pathway [135], dityrosine network [136], and cysteine protease inhibitor in the hemocytes [140] have been analyzed using RNAi. The α2-macroglobulin proteins of I. ricinus, related to vertebrate complement system, were shown to be involved in the phagocytic activity of hemocytes against Gram-negative bacteria [132,133].…”

“…(B.) microplus was implicated as a negative modulator of tick immune response after its silencing greatly reduced the number of bacterial load in the ticks [140]. The role of a defensin from H. longicornis, longicin, in ticks' immune defense against Babesia parasites was demonstrated through RNAi, as exhibited by a higher load of B. gibsoni in the midgut and ovary of longicin-silenced ticks after infestation in an infected host [131].…”