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A tick salivary protein targets cathepsin G and chymase and inhibits host inflammation and platelet aggregation.

ABSTRACT: Platelet aggregation and acute inflammation are key processes in vertebrate defense to a skin injury. Recent studies uncovered the mediation of 2 serine proteases, cathepsin G and chymase, in both mechanisms. Working with a mouse model of acute inflammation, we revealed that an exogenous salivary protein of Ixodes ricinus, the vector of Lyme disease pathogens in Europe, extensively inhibits edema formation and influx of neutrophils in the inflamed tissue. We named this tick salivary gland secreted effector as I ricinus serpin-2 (IRS-2), and we show that it primarily inhibits cathepsin G and chymase, while in higher molar excess, it affects thrombin activity as well. The inhibitory specificity was explained using the crystal structure, determined at a resolution of 1.8 Å. Moreover, we disclosed the ability of IRS-2 to inhibit cathepsin G-induced and thrombin-induced platelet aggregation. For the first time, an ectoparasite protein is shown to exhibit such pharmacological effects and target specificity. The stringent specificity and biological activities of IRS-2 combined with the knowledge of its structure can be the basis for the development of future pharmaceutical applications.

SUBMITTER: Chmelar J

PROVIDER: S-EPMC3031492 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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A tick salivary protein targets cathepsin G and chymase and inhibits host inflammation and platelet aggregation.

Chmelar Jindrich J Oliveira Carlo J CJ Rezacova Pavlina P Francischetti Ivo M B IM Kovarova Zuzana Z Pejler Gunnar G Kopacek Peter P Ribeiro José M C JM Mares Michael M Kopecky Jan J Kotsyfakis Michail M

Blood 20101012 2

Platelet aggregation and acute inflammation are key processes in vertebrate defense to a skin injury. Recent studies uncovered the mediation of 2 serine proteases, cathepsin G and chymase, in both mechanisms. Working with a mouse model of acute inflammation, we revealed that an exogenous salivary protein of Ixodes ricinus, the vector of Lyme disease pathogens in Europe, extensively inhibits edema formation and influx of neutrophils in the inflamed tissue. We named this tick salivary gland secret ...[more]

PMID: 20940421

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Project description:During feeding on vertebrate hosts, ticks secrete saliva composed of a rich cocktail of bioactive molecules modulating host immune responses. Although most of the proteinaceous fraction of tick saliva is of little immunogenicity, repeated feeding of ticks on mammalian hosts may lead to impairment of tick feeding, preventing full engorgement. Here, we challenged rabbits with repeated feeding of both Ixodes ricinus nymphs and adults and observed the formation of specific antibodies against several tick salivary proteins. Repeated feeding of both I. ricinus stages led to a gradual decrease in engorged weights. To identify the salivary antigens, isolated immunoglobulins from repeatedly infested rabbits were utilized for a protein pull-down from the saliva of pilocarpine-treated ticks. Eluted antigens were first identified by peptide mass fingerprinting with the aid of available I. ricinus salivary gland transcriptomes originating from early phases of tick feeding. To increase the authenticity of immunogens identified, we also performed, for the first time, de novo assembly of the sialome from I. ricinus females fed for six days, a timepoint used for pilocarpine-salivation. The most dominant I. ricinus salivary immunogens identified in our study were zinc-dependent metalloproteases of three different families. To corroborate the role of metalloproteases at the tick/host interface, we fed ticks micro-injected with a zinc metalloprotease inhibitor, phosphoramidon, on a rabbit. These ticks clearly failed to initiate feeding and to engorge. However, neither feeding to ticks immune blood of repeatedly infested rabbits, nor phosphoramidon injection into ticks, prevented their engorgement when fed in vitro on an artificial membrane system. These data show that Zn metalloproteases play a decisive role in the success of tick feeding, mediated by complex molecular interactions between the host immune, inflammatory, and hemostatic processes, which are absent in in vitro feeding. This basic concept warrants further investigation and reconsideration of the current strategies towards the development of an effective "anti-tick" vaccine.

Project description:Inflammation and hemostasis are part of the host's first line of defense to tick feeding. These systems are in part serine protease mediated and are tightly controlled by their endogenous inhibitors, in the serpin superfamily (serine protease inhibitors). From this perspective ticks are thought to use serpins to evade host defenses during feeding. The cattle tick Rhipicephalus microplus encodes at least 24 serpins, of which RmS-3, RmS-6, and RmS-17 were previously identified in saliva of this tick. In this study, we screened inhibitor functions of these three saliva serpins against a panel of 16 proteases across the mammalian defense pathway. Our data confirm that Pichia pastoris-expressed rRmS-3, rRmS-6, and rRmS-17 are likely inhibitors of pro-inflammatory and pro-coagulant proteases. We show that rRmS-3 inhibited chymotrypsin and cathepsin G with stoichiometry of inhibition (SI) indices of 1.8 and 2.0, and pancreatic elastase with SI higher than 10. Likewise, rRmS-6 inhibited trypsin with SI of 2.6, chymotrypsin, factor Xa, factor XIa, and plasmin with SI higher than 10, while rRmS-17 inhibited trypsin, cathepsin G, chymotrypsin, plasmin, and factor XIa with SI of 1.6, 2.6, 2.7, 3.4, and 9.0, respectively. Additionally, we observed the formation of irreversible complexes between rRmS-3 and chymotrypsin, rRmS-6/rRmS-17 and trypsin, and rRmS-3/rRmS-17 and cathepsin G, which is consistent with typical mechanism of inhibitory serpins. In blood clotting assays, rRmS-17 delayed plasma clotting by 60 s in recalcification time assay, while rRmS-3 and rRmS-6 did not have any effect. Consistent with inhibitor function profiling data, 2.0 μM rRmS-3 and rRmS-17 inhibited cathepsin G-activated platelet aggregation in a dose-responsive manner by up to 96% and 95% respectively. Of significant interest, polyclonal antibodies blocked inhibitory functions of the three serpins. Also notable, antibodies to Amblyomma americanum, Ixodes scapularis, and Rhipicephalus sanguineus tick saliva proteins cross-reacted with the three R. microplus saliva serpins, suggesting the potential of these proteins as candidates for universal anti-tick vaccines.

Dataset Information

A tick salivary protein targets cathepsin G and chymase and inhibits host inflammation and platelet aggregation.

Publications

A tick salivary protein targets cathepsin G and chymase and inhibits host inflammation and platelet aggregation.

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