A Kazal-type serine protease inhibitor enhances antimicrobial peptide expression in the immune priming of Chinese mitten crab Eriocheir sinensis.

Immune priming confers a sustained, augmented response of innate immune system to a secondary immune stimulation in invertebrate, with involvement of both cellular and humoral immune reactions. Recent evidence indicates that immune effectors, such as antimicrobial peptides, play essential roles in the immune priming. However, the signaling pathways and mechanisms governing their generation remain largely unknown. In the present study, an in vivo immune priming model of Aeromonas hydrophila was established in the crab Eriocheir sinensis. Following a secondary challenge with A. hydrophila, the bacterial load of A. hydrophila in the haemolymph of crabs decreased significantly, and the antibacterial activities of the haemolymph supernatant against A. hydrophila was markedly enhanced. Meanwhile, the mRNA expression level and protein content of Kazal-type serine protease inhibitor-1 (EsKPI-1) in haemolymph was markedly upregulated after the secondary stimulation with A. hydrophila. The recombinant protein of EsKPI-1 (rEsKPI-1) displayed remarkable activity to bind Gram-negative bacteria (A. hydrophila and Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus) in vitro, and the antimicrobial activity to inhibit bacteria growth. Crucially, rEsKPI-1 was found to suppress the extracellular proteases secreted by these pathogens. Following the injection of dsRNA targeting EsKPI-1, there was a significant increase of bacterial load in the haemolymph and a concurrent antibacterial activity reduction of haemolymph against A. hydrophila upon the secondary stimulation with this bacterium. Furthermore, the mRNA transcripts of antimicrobial peptides EsALFs and EsCrustin in the crab haemocytes, as well as those of Toll pathway genes EsTLR-1, EsMyd88 and EsPelle all decreased significantly. The protein contents of EsMyd88 and P-EsDorsal also decreased significantly. These results collectively suggested that the EsKPI-1 played an indispensable role in mediating haemolymph antibacterial responses, particularly in the immune priming induced by A. hydrophila.