A Cysteine Protease Is Critical for Babesia spp. Transmission in Haemaphysalis Ticks

Vector ticks possess a unique system that enables them to digest large amounts of host blood and to transmit various animal and human pathogens, suggesting the existence of evolutionally acquired proteolytic mechanisms. We report here the molecular and reverse genetic characterization of a multifunctional cysteine protease, longipain, from the babesial parasite vector tick Haemaphysalis longicornis. Longipain shares structural similarity with papain-family cysteine proteases obtained from invertebrates and vertebrates. Endogenous longipain was mainly expressed in the midgut epithelium and was specifically localized at lysosomal vacuoles and possibly released into the lumen. Its expression was up-regulated by host blood feeding. Enzymatic functional assays using in vitro and in vivo substrates revealed that longipain hydrolysis occurs over a broad range of pH and temperature. Haemoparasiticidal assays showed that longipain dose-dependently killed tick-borne Babesia parasites, and its babesiacidal effect occurred via specific adherence to the parasite membranes. Disruption of endogenous longipain by RNA interference revealed that longipain is involved in the digestion of the host blood meal. In addition, the knockdown ticks contained an increased number of parasites, suggesting that longipain exerts a killing effect against the midgut-stage Babesia parasites in ticks. Our results suggest that longipain is essential for tick survival, and may have a role in controlling the transmission of tick-transmittable Babesia parasites.