The putative role of Rhipicephalus microplus salivary serpins in the tick-host relationship |
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| Affiliation: | 1. Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil;2. Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA;3. Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil;4. Institute of Biotechnology Genetic Engineering, The University of Agriculture, Peshawar, Khyber Pakhtunkhwa, Pakistan;5. Departamento de Farmacociências, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS, Brazil;6. Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil;1. Faculty of Science, University of South Bohemia in České Budějovice, Budweis, Czech Republic;2. Institute of Parasitology, Biology Center, Czech Academy of Sciences, Budweis, Czech Republic;3. Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA;1. Department of Biomedical Sciences, State University of New York at Albany, Albany, NY, USA;2. Division of Infectious Diseases, Wadsworth Center, New York State Department of Health, Albany, NY, USA;3. Department of Biological Sciences, State University of New York at Albany, Albany, NY, USA;4. Department of Ecology, Evolution, and Environmental Biology, Columbia University, New York, NY, USA;5. Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA;6. Institute of Medical Microbiology and Infection Control, University Hospital of Frankfurt, Frankfurt, Germany;7. Division of Medical Protein Chemistry, Department of Translational Medicine, Lund University, Malmo, Sweden;1. Faculty of Science, University of South Bohemia in České Budějovice, Budweis, Czech Republic;2. Department of Clinical Pathobiochemistry, Technische Universität Dresden, Dresden, Germany;3. Institute of Parasitology, Biology Center, Czech Academy of Sciences, Budweis, Czech Republic;4. Department of Biological Sciences, The University of Southern Mississippi, Hattiesburg, MS, USA;5. Section of Vector Biology, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA;6. Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA |
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| 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. |
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| Keywords: | Immune response Tick saliva Platelet aggregation inhibitor Cathepsin G |
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