A cysteine protease inhibitor (cystatin) from the tick Haemaphysalis longicornis is involved in tick innate immunity

Abstract

Proteins capable of selective and specific inhibition of cysteine protease have been identified as cystatins and are isolated from a variety of microbes and tissues of animals and plants. The physiological function of these proteins have been proposed in regulation of protein turnover and defending against pathogens, as well as in modulating the host's immune response. Genes encoding cystatins have been found in several kinds of ticks, but the function of cystatin in ticks is not understood. We cloned a gene encoding cystatin from the hard tick Haemaphysalis longicornis, and designated it 1-11cyst-2 (H. Iongicornis cystatin-2). The full-length cDNA is 569 bp, encoding a putative 133 amino acids protein with an obvious signal peptide. The cystatin was expressed most in tick midgut and hemocyte. Blood feeding induced a significantly increasing expression in midgut. Real-time PCR confirmed that adult ticks injected with the immuno stimulant lipopolysaccharide (LPS), expressed Hlcyst-2 1.6x more than control ticks injected with phosphate buffered saline. Babesia gibsoni-infected tick larvae expressed Hlcyst-2 1.8x more than uninfected larvae. The recombinant protein also showed a significant growth-inhibitory effect on B. bovis cultured in vitro. These results indicated this cystatin Hlcyst-2 is involved in tick innate immunity.