Characterization of calcineurin A and B genes in the abalone, Haliotis diversicolor, and their immune response role during bacterial infection

Abstract

Calcineurin (CN) is known to be involved in many biological processes, particularly, the immune response mechanism in many invertebrates. In this study, we characterized both HcCNA and HcCNB genes in Haliotis diversicolor, documented their expression in many tissues, and discerned their function as immune responsive genes against Vibrio parahaemolyticus infection. Similar to other mollusk CNs, the HcCNA gene lacked a proline-rich domain and comprised only one isoform of its catalytic unit, in contrast to CNs found in mammals. HcCNB was highly conserved in both sequence and domain architecture. Quantitative PCR and in situ hybridization revealed that the genes were broadly expressed and were not restricted to tissues traditionally associated with immune function. Upon infection of H. diversicolor with V. parahaemolyticus (a bacteria that causes serious disease in crustaceans and mollusks), both HcCNA and HcCNB genes were highly up-regulated at the early phase of bacterial infection. HcCNB was expressed significantly higher than HcCNA in response to bacterial challenge, suggesting its independent or more rapid response to bacterial infection. Together, the two CN genes are unique in their gene structure (particular HcCNA) and distribution in mollusk species and likely function as immune responsive genes along with many other genes that are enhanced in the early phase of V. parahaemolyticus infection in abalone.

Calcineurin subunit B is involved in shell regeneration inHaliotis diversicolor

“…Adult abalone H. diversicolor, about 2 years of age, 55.0 mm in size and 10.0 g in weight, were reared in polyethylene tanks at 23−25 °C with a salinity of 28–30 ppt at Phuket Abalone Farm, Phuket, Thailand and fed daily with fresh kelp as previously described ( Buddawong et al, 2020 ). Animal handling followed the guideline of Animal Care and Use Committee (SCMU-ACUC, Protocol Number MUSC60-040-390), Faculty of Science, Mahidol University.…”

“…qPCR was used to quantify the expression levels of HcCNA and HcCNB . As described previously ( Buddawong et al, 2020 ), 1 µg of the total RNA from mantle was reverse transcribed into cDNA and qPCR was performed in triplicate using a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with Luna Universal qPCR Master Mix (New England Biolabs, Ipswich, MA, USA). The 20 µl reaction mixture contained 0.5 µl (20 ng) of cDNA, 10 µl of Luna Universal qPCR mix with Hot Start Taq DNA Polymerase, 0.5 µl of 10 µM of each primer (for primer details see Table 1 ), and 8.5 µl of PCR grade water.…”

“…To express the recombinant HcCNA and HcCNB in E.coli , the coding regions of HcCNA and HcCNB obtained from the previous study ( Buddawong et al, 2020 ) were amplified by PCR using 2 pairs of primers (expressed CNA-F and expressed CNA-R for HcCNA and expressed CNB-F and expressed CNB-R for HcCNB) which contained the positions of NcoI/BamHI digestion and NdeI/BamHI digestion for HcCNA and HcCNB, respectively. The PCR products were purified with FavorPrep GEL/PCR purification kit (Favorgen, Ping-Tung, Taiwan) and digested with NcoI/BamHI and NdeI/BamHI for HcCNA and HcCNB, respectively.…”

“…The upregulation of CNB level over CNA has been reported in cancer tissues ( Wei, Zhang & Wang, 1993 ), again suggesting an independent role for CNB, and the administration of recombinant human CNB showed a high anti-cancer potency with an unclear molecular mechanism ( Jin et al, 2005 ). Recently, we also found that CNB has a more pronounced immuno-protective response than CNA in the abalone Haliotis diversicolor (a gastropod mollusk) in response to foreign microbial challenge ( Buddawong et al, 2020 ). In this previous study we also noted constitutive expression of both HcCNA and HcCNB in the shell secreting epithelium, this, and the proposed role for calcineurin in shell formation in the bivalve Pinctada fucata ( Li et al, 2010 ), prompted us to investigate the potential roles for calcineurin subunits in biomineralization in H. diversicolor .…”