Initiator and executioner caspases in salivary gland apoptosis of Rhipicephalus haemaphysaloides

Abstract

Background: Apoptosis is fundamental in maintaining cell balance in multicellular organisms, and caspases play a crucial role in apoptosis pathways. It is reported that apoptosis plays an important role in tick salivary gland degeneration. Several different caspases have been found in ticks, but the interactions between them are currently unknown. Here, we report three new caspases, isolated from the salivary glands of the tick Rhipicephalus haemaphysaloides. Methods: The full-length cDNA of the RhCaspases 7, 8 and 9 genes were obtained by transcriptome, and RhCaspases 7, 8 and 9 were expressed in E. coli; after protein purification and immunization in mice, specific polyclonal antibodies (PcAb) were created in response to the recombinant protein. Reverse-transcription quantitative PCR (RT-qPCR) and western blot were used to detect the existence of RhCaspases 7, 8 and 9 in ticks. TUNEL assays were used to determine the apoptosis level in salivary glands at different feeding times after gene silencing. The interaction between RhCaspases 7, 8 and 9 were identified by co-transfection assays. Results: The transcription of apoptosis-related genes in R. haemaphysaloides salivary glands increased significantly after tick engorgement. Three caspase-like molecules containing conserved caspase domains were identified and named RhCaspases 7, 8 and 9. RhCaspase8 and RhCaspase9 contain a long pro-domain at their N-terminals. An RT-qPCR assay demonstrated that the transcription of these three caspase genes increased significantly during the engorged periods of the tick developmental stages (engorged larval, nymph, and adult female ticks). Transcriptional levels of RhCaspases 7, 8 and 9 in salivary glands increased more significantly than other tissues post-engorgement. RhCaspase9-RNAi treatment significantly inhibited tick feeding. In contrast, knockdown of RhCaspase7 and RhCas-pase8 had no influence on tick feeding. Compared to the control group, apoptosis levels were significantly reduced after interfering with RhCaspase 7, 8 and 9 expressions. Co-transfection assays showed RhCaspase7 was cleaved by RhCaspases 8 and 9, demonstrating that RhCaspases 8 and 9 are initiator caspases and RhCaspase7 is an executioner caspase. Conclusions: To the best of our knowledge, this is the first study to identify initiator and executioner caspases in ticks, confirm the interaction among them, and associate caspase activation with tick salivary gland degeneration.

Identification of the Bcl-2 and Bax Homologs From the Tick Rhipicephalus Haemaphysaloides and Their Function in the Degeneration of Tick Salivary Glands

“…For phylogenetic analysis, the alignment of the sequences was performed using the MUSCLE algorithm [31] and inferred using the Maximum likelihood method with the default settings in MEGA X software [32]. Bootstrap support values were estimated using 500 bootstrap replicates [28].…”

“…Speci c RhBcl-2 and RhBax primers (Table S2) were designed in a pET-30a or PGEX-4T-1 vector. The RhBcl-2 and RhBax ampli ed-PCR products were puri ed and digested with BamHI, EcoRI, or XhoI (New England Biolabs, USA) and ligated into pET-28a or PGEX-4T-1(Invitrogen) using In-Fusion HD Cloning Kits (Takara Clontech, Mountain View, CA, USA) [28]. These recombinants were transformed and expressed in Escherichia coli (E. coli) BL21 (DE3) strain (TIANGEN, Beijing, China).…”

“…HEK 293 cells were maintained in Dulbecco's Modi ed Eagle Medium (DMEM, Gibco), supplemented with 8% heat-inactivated fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel) and 1% penicillin (Gibco) at 37°C [28].…”

“…Transfection, using Lipofectamine™ 3000 Transfection Reagent (Invitrogen), was performed according to manufacturer protocol with a DNA to Lipofectamine ratio of 1: 2 w/v. The HEK 293 cells were transformed with 2.5 µg/well of plasmid or co-transfected with the equivalent amount of two different plasmids in 6-well plates [28].…”

“…The membranes were rinsed ve times by TBST. Images were captured by ChemiDoc Touch (Bio-rad, Hercules, CA, USA) [28].…”