The hard tick Ixodes ricinus is a vector of Lyme disease and tick-borne encephalitis. Host blood protein digestion, essential for tick development and reproduction, occurs in tick midgut digestive cells driven by cathepsin proteases. Little is known about the regulation of the digestive proteolytic machinery of I. ricinus. Here we characterize a novel cystatin-type protease inhibitor, mialostatin, from the I. ricinus midgut. Blood feeding rapidly induced mialostatin expression in the gut, which continued after tick detachment. Recombinant mialostatin inhibited a number of I. ricinus digestive cysteine cathepsins, with the greatest potency observed against cathepsin L isoforms, with which it co-localized in midgut digestive cells. The crystal structure of mialostatin was determined at 1.55 Å to explain its unique inhibitory specificity. Finally, mialostatin effectively blocked in vitro proteolysis of blood proteins by midgut cysteine cathepsins. Mialostatin is likely to be involved in the regulation of gut-associated proteolytic pathways, making midgut cystatins promising targets for tick control strategies.

Department of Biochemistry Faculty of Science Charles University Hlavova 2030 8 12800 Prague Czech Republic

Department of Medical Biology Faculty of Science University of South Bohemia in České Budějovice Branišovská 1760c 37005 České Budějovice Czech Republic

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Flemingovo n 2 16610 Praha Czech Republic

Institute of Parasitology Biology Centre Academy of Sciences of the Czech Republic Branišovská 1160 31 37005 České Budějovice Czech Republic

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Editorial: Special Issue on the "Molecular Biology of Disease Vectors"