title: Effective inhibition of thrombin by Rhipicephalus microplus serpin-15 (RmS-15) obtained in the yeast Pichia pastoris.
abstract: The cattle tick (Rhipicephalus microplus) affects cattle industries in tropical and subtropical countries because it is the vector of babesiosis and anaplasmosis which constitutes a threat to the health of cattle. During blooding feeding, ticks secrete saliva containing a complex of bioactive molecules into the injured site to evade host's defensive responses. Serine protease inhibitors (serpins) are important anti-haemostatic molecules present in tick saliva that are necessary for a successful blood feeding. Several serpin sequences have been reported in R. microplus but there is a gap of information about their functions during host-parasite interactions. In this study, the RmS-15 expressed in the yeast Pichia pastoris was characterised using kinetic assays and in vitro analysis. The inhibitory enzymatic assays conducted showed that RmS-15 is a physiological inhibitor of thrombin with a stoichiometric inhibition (SI) of 1.5 and high inhibition affinity with ka=9.3±0.5×104M(-1)s(-1). RmS-15 delayed the clotting of plasma in a dose-dependent manner as determined in a recalcification time assay. Significant elevated ELISA titres were observed in tick resistant and susceptible cattle on day 28 after the tick infestation (p<0.001). This data suggests direct contact of RmS-15 with the immune system of the host at the tick-feeding site. The present study contributed to the understanding of the biological functions of R. microplus serpins during host-parasite interactions which contributes to the design of future innovative methods for tick control.
authors.affiliation: The University of Queensland, Queensland Alliance for Agriculture & Food Innovation, Qld, Australia.
authors.forename: Tao
authors.initials: T
authors.lastname: Xu
authors.affiliation: The University of Queensland, Queensland Alliance for Agriculture & Food Innovation, Qld, Australia; Murdoch University, Centre for Comparative Genomics, Perth, Western Australia 6150, Australia.
authors.forename: Ala
authors.initials: A
authors.lastname: Lew-Tabor
authors.affiliation: The University of Queensland, Queensland Alliance for Agriculture & Food Innovation, Qld, Australia. Electronic address: m.rodriguezvalle@uq.edu.au.
authors.forename: Manuel
authors.initials: M
authors.lastname: Rodriguez-Valle
mesh_terms: D000818:Animals; D000991:Antithrombins / Q000378:metabolism / Q000494:pharmacology*; D005786:Gene Expression Regulation; D010843:Pichia / Q000378:metabolism*; D048494:Rhipicephalus / Q000378:metabolism*; D015843:Serpins / Q000378:metabolism / Q000494:pharmacology*; D013917:Thrombin / Q000378:metabolism*
chemical_list: D000991:Antithrombins; D015843:Serpins; D013917:Thrombin
keywords: Anticoagulant; Cattle ticks; R. microplus; Serpins; Thrombin inhibitors