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Published on: December 21, 2019
Rhipicephalus microplus serine protease inhibitor family: annotation, expression and functional characterisation
Manuel Rodriguez-Valle1, Tao Xu2, Sebastian Kurscheid3
1The 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.
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).
Conclusion:
This study constitutes an important contribution and improvement to the knowledge about the physiologic role of R. microplus serpins during the host-tick interaction.
Insights
This study identified new Rhipicephalus (Boophilus) microplus serpins (RmS-19 to RmS-22) and analyzed their expression and function. These tick serine protease inhibitors play crucial roles in host-tick interactions.
Area of Science:
- Molecular Biology
- Bioinformatics
- Parasitology
Background:
- Rhipicephalus (Boophilus) microplus utilizes secreted proteins, including serine protease inhibitors (serpins), to evade host hemostasis.
- Serpins are a significant family of protease inhibitors in Ixodidae ticks, modulating essential proteases in various physiological processes.
Purpose of the Study:
- To identify and characterize novel serpin sequences in Rhipicephalus (Boophilus) microplus.
- To investigate the expression patterns and functional roles of identified R. microplus serpins in host-tick interactions.
Main Methods:
- Bioinformatic analysis of R. microplus serpin sequences using the Yabi environment and various transcriptome databases.
- Semi-quantitative PCR to determine gene expression across different tick tissues and developmental stages.
- Cloning and expression of selected R. microplus serpins in Pichia pastoris for protease inhibition assays.
Main Results:
- Four new R. microplus serpins (RmS-19 to RmS-22) were identified, showing high sequence conservation.
- Ubiquitous expression of most identified serpins was observed, with specific expression patterns for RmS-6 and RmS-14.
- Expressed recombinant serpins demonstrated specific inhibition of proteases including Chymotrypsin, Elastase, and Thrombin.
Conclusions:
- The study expands the understanding of R. microplus serpin diversity and their physiological roles.
- Findings contribute to knowledge on how R. microplus serpins mediate host-tick interactions.
