Identification, Expression and Preliminary Antimicrobial Mechanism of Liver-expressed Antimicrobial Peptide 2 Gene in Black Scraper (Thamnaconus modestus)
HU Yi-Fan, WEI Bo-Jue, ZHANG Yi-Rong, LI Chang-Hong*, CHEN Jiong
Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315832, China
Abstract：Liver-expressed antimicrobial peptide 2 (LEAP-2) is a cationic antimicrobial peptide that plays a crucial role in the innate immunity of animals. The cDNA sequences of TmLEAP-2A and TmLEAP-2C were identified through transcriptome sequencing in black scraper (Thamnaconus modestus) in this study. Sequence analysis indicated that TmLEAP-2A and TmLEAP-2C encoded 103 and 79 amino acids, respectively, with molecular weights of 11.18 and 9.14 kD. TmLEAP-2A and TmLEAP-2C exhibited the highest amino acid homology with LEAP-2A and LEAP-2C from the large yellow croaker (Larimichthys crocea), at 72.8% and 62.5% respectively. Phylogenetic tree analysis showed that TmLEAP-2A and TmLEAP-2C had the closest evolutionary correlation with LEAP-2A and LEAP-2C in large yellow croaker respectively. qPCR analysis showed that both TmLEAP-2A and TmLEAP-2C were predominately expressed in the liver. Further, their expression levels were significantly induced by infection with Vibrio alginolyticus in liver of black scraper. TmLEAP-2A mature peptide (TmLEAP-2AP46) only exhibited inhibitory activity against V. vulnificus, while TmLEAP-2C mature peptide (TmLEAP-2CP39) exhibited inhibitory activity against both V. alginolyticus and V. vulnificus. Further research demonstrated that TmLEAP-2CP39 exhibited a hydrolytic effect on the genome of V. alginolyticus. Following incubation with TmLEAP-2CP39, V. alginolyticus showed increased uptake of propidium iodide, indicating a significant reduction in the proportion of viable cells. Scanning electron microscopy (SEM) observations revealed that treatment with TmLEAP-2CP39 resulted in altered morphology of V. alginolyticus, characterized by surface wrinkles and the presence of depressions or holes on most bacterial cells. Additionally, transmission electron microscopy (TEM) analysis revealed that, after treatment with TmLEAP-2CP39, the cell membrane of V. alginolyticus exhibited pronounced wrinkling, significant separation from the cytoplasm, rupturing of the membrane, and notable leakage of cellular contents. Overall, TmLEAP-2A and TmLEAP-2C were found to be closely associated with V. alginolyticus infection; however, only TmLEAP-2C demonstrated antibacterial activity against V. alginolyticus in vitro. TmLEAP-2C exerted direct antibacterial effects by disrupting the integrity of the bacterial cell membrane and hydrolyzing genomic DNA (gDNA) within V. alginolyticus cells. These findings not only enhance the understanding of fish antimicrobial peptides but also provide a theoretical foundation for the research and development of fish-derived antimicrobial peptide drugs.
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