Carboxymethyl chitosan nanoparticles loaded with bioactive protein CiCXCL20a effectively prevent bacterial disease in grass carp (Ctenopharyngodon idella)

Oral antimicrobial proteins have been widely studied for the prevention of bacterial diseases in aquaculture. However, the shortcomings of antimicrobial proteins that are easily degraded in vivo limits the application. To address these challenges, we expressed the antimicrobial chemokine Ctenopharyngodon idella CXCL20a (CiCXCL20a) by Pichia pastoris and loaded it by carboxymethyl chitosan (CMCS). We used dynamic light scattering (DLS) and transmission electron microscopy (TEM) for characterization and confirmation, obtained stable and slow-release nanoparticle CMCS-20a. In vitro experiments, CMCS-20a can effectively kill bacteria by destroying the bacterial membrane and binding DNA, and reduce the influence of enzyme, salt ion and serum on antibacterial activity of CiCXCL20a. Furthermore, CMCS-20a was almost nontoxic to piscine cells and bound to bacteria when eukaryotic cells coexist with bacteria. More importantly, oral CMCS-20a (mixed with feed) can increase the activities of total superoxide dismutase (SOD), lysozyme (LZM), complement 3 (C3) in sera. After Aeromonas hydrophila infection, oral CMCS-20a enhances grass carp survival rate. To uncover the protective mechanism, we investigated the inflammatory response, bacterial loads and tissue injury. The results indicated that oral CMCS-20a can efficiently regulate inflammation responses by swiftly upregulating and recovering mRNA expressions of inflammation-related genes (IL-1 beta, TNF-alpha, IL-2, IFN-gamma 2) in head kidney tissue, significantly reduce bacterial burdens in intestine, head kidney, hepatopancreas, spleen, blood tissues, and obviously alleviate tissue lesions in spleen and intestine tissues. In conclusion, nanoparticle CMCS-20a has a good biosafety and can effectively prevent bacterial disease in grass carp.