title: Rhipicephalus microplus serine protease inhibitor family: annotation, expression and functional characterisation assessment.
abstract: BACKGROUND
  Rhipicephalus (Boophilus) microplus evades the host's haemostatic system through a complex protein array secreted into tick saliva. Serine protease inhibitors (serpins) conform an important component of saliva which are represented by a large protease inhibitor family in Ixodidae. These secreted and non-secreted inhibitors modulate diverse and essential proteases involved in different physiological processes.
  
  
  METHODS
  The identification of R. microplus serpin sequences was performed through a web-based bioinformatics environment called Yabi. The database search was conducted on BmiGi V1, BmiGi V2.1, five SSH libraries, Australian tick transcriptome libraries and RmiTR V1 using bioinformatics methods. Semi quantitative PCR was carried out using different adult tissues and tick development stages. The cDNA of four identified R. microplus serpins were cloned and expressed in Pichia pastoris in order to determine biological targets of these serpins utilising protease inhibition assays.
  
  
  RESULTS
  A total of four out of twenty-two serpins identified in our analysis are new R. microplus serpins which were named as RmS-19 to RmS-22. The analyses of DNA and predicted amino acid sequences showed high conservation of the R. microplus serpin sequences. The expression data suggested ubiquitous expression of RmS except for RmS-6 and RmS-14 that were expressed only in nymphs and adult female ovaries, respectively. RmS-19, and -20 were expressed in all tissues samples analysed showing their important role in both parasitic and non-parasitic stages of R. microplus development. RmS-21 was not detected in ovaries and RmS-22 was not identified in ovary and nymph samples but were expressed in the rest of the samples analysed. A total of four expressed recombinant serpins showed protease specific inhibition for Chymotrypsin (RmS-1 and RmS-6), Chymotrypsin / Elastase (RmS-3) and Thrombin (RmS-15).
  
  
  CONCLUSIONS
  This study constitutes an important contribution and improvement to the knowledge about the physiologic role of R. microplus serpins during the host-tick interaction.
authors.affiliation: The University of Queensland, Queensland Alliance for Agriculture & Food Innovation, Queensland Biosciences Precinct, 306 Carmody Rd, St. Lucia Qld, 4072, Australia. m.rodriguezvalle@uq.edu.au.
authors.forename: Manuel
authors.initials: M
authors.lastname: Rodriguez-Valle
authors.affiliation: The University of Queensland, Queensland Alliance for Agriculture & Food Innovation, Queensland Biosciences Precinct, 306 Carmody Rd, St. Lucia Qld, 4072, Australia. tao.xu@uqconnect.edu.au.
authors.forename: Tao
authors.initials: T
authors.lastname: Xu
authors.affiliation: University Hospital Lausanne, Lausanne, VD, Switzerland. sebastian.kurscheid@gmail.com.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Kurscheid
authors.affiliation: The University of Queensland, Queensland Alliance for Agriculture & Food Innovation, Queensland Biosciences Precinct, 306 Carmody Rd, St. Lucia Qld, 4072, Australia. a.lewtabor@uq.edu.au.|Murdoch University, Centre for Comparative Genomics, Perth, Western Australia, 6150, Australia. a.lewtabor@uq.edu.au.
authors.forename: Ala E
authors.initials: AE
authors.lastname: Lew-Tabor
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology; D009758:Nymph; D010053:Ovary; D010447:Peptide Hydrolases / Q000378:metabolism; D048494:Rhipicephalus / Q000235:genetics / Q000378:metabolism*; D012463:Saliva / Q000737:chemistry*; D015842:Serine Proteinase Inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D015842:Serine Proteinase Inhibitors; D004247:DNA; D010447:Peptide Hydrolases