Characterization and expression analysis of Calmodulin (CaM) in orange-spotted grouper (Epinephelus coioides) in response to Vibrio alginolyticus challenge

Abstract

Vibrio alginolyticus containing the highly toxic extracellular product is one of the most serious threats to grouper survival and its minimum lethal dose is approximately 500 CFU/g fish body weight in grouper. To study the toxic effects of V. alginolyticus on the immune system in teleost, Calmodulin (CaM), an important molecular indicator gene, was cloned from the orange-spotted grouper (Epinephelus coioides). The full-length Ec-CaM consisted of a 5'-UTR of 103 bp, an ORF of 450 bp and a 3'-UTR of 104 bp. The Ec-CaM gene encoded a protein of 149 amino acids with an estimated molecular mass of 16.4 kDa and a predicted isoelectric point of 3.93. The deduced amino acid sequence showed that Ec-CaM contained four highly conserved EF-hand domains known to be critical for the function of CaM. Ec-CaM was widely expressed and the highest expression level was observed in liver. Following V. alginolyticus challenge, a sharp increase level of respiratory burst activity and apoptosis ratio were observed. Further analyses of CaM expression and p53 expression in liver, kidney and spleen by qRT-PCR demonstrated that the up-regulated expression of CaM and p53 were observed in the vibrio challenge group. Western blotting analysis confirmed that the Ec-CaM protein was strongly induced in liver at 12 h post-injection, while a sharp increase of p53 protein expression was observed at 24 h post-injection. These results showed CaM expression serving as a potential molecular indicator may help to assess the toxicological effects of V. alginolyticus on the ROS generation and apoptotic process in grouper.

Calmodulin Gene of Blunt Snout Bream (Megalobrama amblycephala): Molecular Characterization and Differential Expression after Aeromonas hydrophila and Cadmium Challenges

“…The tissue expression of MaCalm mRNA was similar to that of Calm in the hybrid F 1 of Acanthopagrus schlegelii male × Pagrus major female [16] and Drosophila melanogaster [38]. Although the structure of Calm is similar across species, some differences in Calm tissue distribution are found among different animal species [14,15,39,40]. The constitutive distribution and diversified expression patterns of MaCalm in different tissues are likely related to the multiple functions of Calm.…”

“…In this study, the significant downregulation of MaCalm in the kidney was detected after A. hydrophila infection, suggesting MaCalm played pivotal roles in fish defense against the invasion of pathogens. However, a notable upregulation of Calm in the kidney was observed after Vibrio alginolyticus infection in Epinephelus coioides [39], suggesting that the expression patterns of Calm varied depending on the animal species and microbial strains. Unlike in the kidney, MaCalm mRNA expression in the liver increased notably at 12 hpi after A. hydrophila infection, which might respond to the microbial challenge and neutralize the adverse effects of the Aeromonas infection [39].…”

“…However, a notable upregulation of Calm in the kidney was observed after Vibrio alginolyticus infection in Epinephelus coioides [39], suggesting that the expression patterns of Calm varied depending on the animal species and microbial strains. Unlike in the kidney, MaCalm mRNA expression in the liver increased notably at 12 hpi after A. hydrophila infection, which might respond to the microbial challenge and neutralize the adverse effects of the Aeromonas infection [39]. Intriguingly, several pieces of research show that Calm could act as a co-factor of virulence factors of bacteria to prevent innate immune activation [20].…”

“…These results indicate that HSP70 transcripts could respond differentially to biotic and abiotic stressors. Calm is considered as one of the most important molecular biomarkers in teleost fishes under chronic stressors [39]. Therefore, the combination of the MaCalm and HSP70 expression level in the liver could be regarded as the potential biomarkers of Cd-induced toxicity.…”