Abstract
Marine organisms are an outstanding source of molecules with wide biological activities. In this research, we investigated the antimicrobial protein haemocyanin isolated from the haemolymph of flower crab (Portunus pelagicus) using gel filtration chromatography. Purified P. pelagicus haemocyanin (Pp-Hc) was subjected to SDS-PAGE analysis and the fraction containing Pp-Hc ran as a distinct band, its molecular weight was 78 kDa. The Pp-Hc was characterized through HPLC, XRD, CD and FTIR analyses. The antibacterial potential of Pp-Hc was evaluated against five Gram-positive and ten Gram-negative bacteria through the agar well diffusion method, which clearly revealed the high bactericidal activity of Pp-Hc. Furthermore, Pp-Hc was studied for its antibiofilm activity against five biofilm-forming Gram-negative bacteria, i.e., Vibrio alginolyticus, Vibrio harveyi, Vibrio parahaemolyticus, Pseudomonas aeruginosa, and Proteus vulgaris. Confocal laser scanning microscopy (CLSM) and light microscopy evidenced significant antibiofilm properties of Pp-Hc at 100 mu g/ml concentration. Based on exopolysaccharide (EPS) quantification and cell surface hydrophobicity (CSH) index, the antibiofilm action of Pp-Hc was linked to its ability to reduce EPS production and increase cell surface hydrophobicity. The pathogen recognition mechanism of purified Pp-Hc showed a wide spectrum of binding activities, leading to high toxicity against both Gram-positive and Gram-negative bacterial species. Furthermore, the purified Pp-Hc showed high antifungal activity against the pathogen Candida albicans. Haemocyanin is not only involved in transporting oxygen, it has a relevant function in the flower crab immune system. This work adds useful knowledge for aquaculture concerns dealing with microbial pathogen control. Overall, our results outlined the high potential of purified Pp-Hc as a starting material to develop new and effective antimicrobial drugs for aquaculture purposes.