Abstract
Tick saliva is a rich source of antihemostatic, anti-inflammatory, and immunomodulatory molecules that actively help the tick to finish its blood meal. Moreover, these molecules facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-8, a salivary serpin from the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Iripin-8 displayed blood-meal-induced mRNA expression that peaked in nymphs and the salivary glands of adult females. Iripin-8 inhibited multiple proteases involved in blood coagulation and blocked the intrinsic and common pathways of the coagulation cascade in vitro. Moreover, Iripin-8 inhibited erythrocyte lysis by complement, and Iripin-8 knockdown by RNA interference in tick nymphs delayed the feeding time. Finally, we resolved the crystal structure of Iripin-8 at 1.89 Å resolution to reveal an unusually long and rigid reactive center loop that is conserved in several tick species. The P1 Arg residue is held in place distant from the serpin body by a conserved poly-Pro element on the P′ side. Several PEG molecules bind to Iripin-8, including one in a deep cavity, perhaps indicating the presence of a small-molecule binding site. This is the first crystal structure of a tick serpin in the native state, and Iripin-8 is a tick serpin with a conserved reactive center loop that possesses antihemostatic activity that may mediate interference with host innate immunity.
Highlights
Ticks are blood-feeding ectoparasites and vectors of human pathogens, including agents of Lyme disease and tick-borne encephalitis
Similar to other characterized tick salivary serpins [13], we found that Iripin-8 can modulate host complement and coagulation cascades to facilitate tick feeding [46]
Iripin-8 has an unusually long, exposed, and rigid reactive center loop (RCL), with an Arg in its P1 position. This potentially enables it to inhibit a range of proteases, as the RCL can interact independently from the body of the serpin molecule
Summary
Ticks are blood-feeding ectoparasites and vectors of human pathogens, including agents of Lyme disease and tick-borne encephalitis. Blood coagulation is a cascade driven by serine proteases that leads to the production of a fibrin clot. It can be initiated via the extrinsic or intrinsic pathway [9]. Small C3a and C5a subunits promote inflammation by recruiting immune cells to the site of injury [11] Both processes, coagulation and complement, are detrimental to feeding ticks, so their saliva contains many anticoagulant and anticomplement molecules, often belonging to the group of serine protease inhibitors (serpins) [13,14,15,16]. Over 20 tick salivary serpins have been functionally characterized with described effects on coagulation or immunity [13]. I. ricinus as IRS-8 [30], and it can be found among transcripts of other tick species in which the serpins have not yet been functionally characterized
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