The genetic characteristics of Pahepcidin1 and Pahepcidin2 in silver pomfret (Pampus argenteus) and their antibacterial functions in innate immunity.

Abstract

Hepcidins are a class of cysteine-rich antimicrobial peptides that exert significant impacts in response to a variety of pathogens. In this study, two hepcidins were identified from silver pomfret (Pampus argenteus), namely Pahepcidin1 and Pahepcidin2. Our findings revealed that Pahepcidin1 consists of a 267 open reading frame (ORF) encoding 91 amino acids, while Pahepcidin2 consists of 246 bp ORF encoding 81 amino acids. Sequence comparison between Pahepcidins and other homologous genes demonstrated the presence of eight conserved cysteines in the C-terminus region of Pahepcidin1 and six cysteine residues in the C-terminus region of Pahepcidin2. Phylogenetic analysis indicated that both Pahepcidin1 and Pahepcidin2 clustered with other fish homologues, respectively. The results of tissue-specific expression analysis showed that Pahepcidins exhibited the highest expression level in the liver; moreover, the expression level of Pahepcidin1 in silver pomfret liver cells (PaL cells) was also significantly increased after stimulation with lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (poly I:C), and Staphylococcus aureus (S. aureus). Meanwhile, this study verified the antibacterial function of recombinant Pahepcidin1, and the results indicated that it possessed broad antibacterial activity against both Gram-positive bacteria (S. aureus) and Gram-negative bacteria (Edwardsiella tarda). In addition, the overexpression of Pahepcidin1 could significantly alter the transcriptional levels of key genes in the NF-κB pathway and downstream immune-related genes. In vivo validation via Vibrio parahaemolyticus challenge showed that Pahepcidin1 expression in silver pomfret liver was significantly upregulated at 3 h and peaked at 6 h, then downregulated to below the baseline from 12 h to 48 h. These results suggested that Pahepcidin1 might play an essential role in the innate immune system modulation of silver pomfret, providing valuable insights into understanding the antimicrobial immune mechanism of this species.