Abstract
Piscidins are antimicrobial peptides (AMPs) that play important roles in helping fish resist pathogenic infections. Through comparisons of tilapia EST clones, the coding sequences of five piscidin-like AMPs (named TP1∼5) of Nile tilapia, Oreochromis niloticus, were determined. The complete piscidin coding sequences of TP1, -2, -3, -4, and -5 were respectively composed of 207, 234, 231, 270, and 195 bases, and each contained a translated region of 68, 77, 76, 89, and 64 amino acids. The tissue-specific, Vibrio vulnificus stimulation-specific, and Streptococcus agalactiae stimulation-specific expressions of TP2, -3, and -4 mRNA were determined by a comparative RT-PCR. Results of the tissue distribution analysis revealed high expression levels of TP2 mRNA in the skin, head kidneys, liver, and spleen. To study bacterial stimulation, S. agalactiae (SA47) was injected, and the TP4 transcript was upregulated by >13-fold (compared to the wild-type (WT) control, without injection) and was 60-fold upregulated (compared to the WT control, without injection) 24 h after the S. agalactiae (SA47) injection in the spleen and gills. Synthesized TP3 and TP4 peptides showed antimicrobial activities against several bacteria in this study, while the synthesized TP1, -2, and -5 peptides did not. The synthesized TP2, -3, and -4 peptides showed hemolytic activities and synthesized TP3 and TP4 peptides inhibited tilapia ovary cell proliferation with a dose-dependent effect. In summary, the amphiphilic α-helical cationic peptides of TP3 and TP4 may represent novel and potential antimicrobial agents for further peptide drug development.
Highlights
Antimicrobial peptides (AMPs) are cationic peptides that play important roles in innate immunity
The blast analysis showed that the sequence similarity of tilapia piscidin 1 (TP1) with TP2 was 51%, that of TP1 with TP3 was 40%, that of TP1 with TP4 was 61%, that of TP1 with TP5 was 37%, that of TP2 with TP3 was 48%, that of TP2 with TP4 was 52%, that of TP2 with TP5 was 26%, that of TP3 with TP4 was 63%, that of TP3 with TP5 was 48%, and that of TP4 with TP5 was 52%. These sequences were input for NCBI blast analyses, and results suggested that TP1 was related to the O. niloticus pleurocidin-like peptide, WF4-like, with an identity of 43%
To study whether bacterial infection had any effects on piscidin expressions, we showed levels of Nile tilapia piscidin mRNA expressions in different tissues after V. vulnificus (204) stimulation (Fig. 4b) and S. agalactiae (SA47) stimulation (Fig. 4b) at different times (24 and 48 h)
Summary
Antimicrobial peptides (AMPs) are cationic peptides that play important roles in innate immunity. The synthetic piscidin 2 presented potent activity against the water mould Saprolegnia, and the minimum oomyceticidal concentration (MOC 100) ranged 12.5,25 mg/ml [20] Those results suggest that piscidins are similar to other AMPs which possess antimicrobial activities [21]. Fish piscidins possess antimicrobial activities in vitro, and in fish, are stored in granules of phagocytic granulocytes and are delivered to pathogen-containing phagosomes upon phagocytosis [22], which suggests that fish may use piscidins as antimicrobial agents in vivo to kill invading pathogens Another interesting research result suggested that positively selected sites identified in the Atlantic cod (Gadus morhua L.) piscidin gene that codes for the mature peptide are associated with the adaptation of piscidins to pathogens and may be involved in protecting the host against rapidly evolving pathogens [23]. These research results mentioned above support cod piscidins being important immune-related genes in the cod innate immune system and playing multifunctional roles in Atlantic cod following their structural diversification [25]
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