title: The putative role of Rhipicephalus microplus salivary serpins in the tick-host relationship.
abstract: 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.
authors: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA.
authors: Lucas
authors: L
authors: Tirloni
authors: Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA.
authors: Tae Kwon
authors: TK
authors: Kim
authors: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors: Mariana Loner
authors: ML
authors: Coutinho
authors: Institute of Biotechnology Genetic Engineering, The University of Agriculture, Peshawar, Khyber Pakhtunkhwa, Pakistan.
authors: Abid
authors: A
authors: Ali
authors: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; Departamento de Farmacociências, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS, Brazil.
authors: Adriana
authors: A
authors: Seixas
authors: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors: Carlos
authors: C
authors: Termignoni
authors: Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA.
authors: Albert
authors: A
authors: Mulenga
authors: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. Electronic address: itabajara.vaz@ufrgs.br.
authors: Itabajara
authors: I
authors: da Silva Vaz
mesh_terms: D000818:Animals; D060829:Arthropod Proteins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002418:Cattle Diseases / Q000469:parasitology*; D005260:Female; D006790:Host-Parasite Interactions; D008297:Male; D005810:Multigene Family; D048494:Rhipicephalus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D012463:Saliva / Q000201:enzymology; D015843:Serpins / Q000235:genetics / Q000378:metabolism*; D013984:Tick Infestations / Q000469:parasitology / Q000662:veterinary*
chemical_list: D060829:Arthropod Proteins; D015843:Serpins
keywords: Cathepsin G; Immune response; Platelet aggregation inhibitor; Tick saliva