Piscidin-like antimicrobial peptide precursor

Piscidins, important components of the innate (nonspecific) immunity system in fish, have potent, broad-spectrum antimicrobial and antiparasitic activities. In this study, we reported a novel antimicrobial cationic peptide from Pseudosciaena crocea. Although this peptide exhibited a genomic (3 exons and 2 introns) and propeptide (signal peptide, mature peptide and prodomain) organization, conserved signal peptide (22 amino acids) and consensus motif I-X5-H-X4-I-H identical to the reported fish piscidins, Pc-pis showed a relatively low overall conservation with other known piscidins, which was obviously embodied in the amino acid composition of the peptide. Pc-pis is strikingly rich in glycine residues (27.3%), which disrupted the amphipathic structure of the peptide. Relative quantitative real-time PCR revealed that Pc-pis is a typically gill-expressed peptide. The sequence analysis, structural features and tissue distribution suggested that Pc-pis was genetically related to the piscidins family and might be a novel piscidin-like antimicrobial peptide. Quantitative PCR analysis revealed that the expression of Pc-pis in the spleen, head-kidney, liver, intestine, skin and gill could be regulated during Cryptocaryon irritans infection and post C. irritans falling off, implicating a role for Pc-pis in immune defense against C. irritans and secondary bacterial infections. Synthetic Pc-pis exhibited broad-spectrum activity against bacteria, fungi and C. irritans in parasitic stages. These results provided the first evidence of piscidins antiparasitic activity against marine fish ectoparasites C. irritants trophonts and further indicated that Pc-pis might be an important component of the P. crocea innate immune system against C. irritans and secondary bacterial infections. Thus, these data provided new insights into P. crocea innate immunity against external protozoan parasite and microbial infections and facilitate the evaluation of Pc-pis as a therapeutic agent against pathogen invasion.

The piscidin family comprises a group of antimicrobial peptides (AMPs) that are vital components of teleost innate immunity. Piscidins protect the host from pathogens, through multifaceted roles as immunomodulators and anti-infective peptides. The present study reports the identification, and characterization of a putative piscidin homolog, Of-Pis1, from rock bream (Oplegnathus fasciatus). A combined genomic and transcriptomic approach revealed that the Of-Pis1 gene comprises 1396 nucleotides (nt), four exons, and three introns. The cDNA with the 213 nt open reading frame encoded a 70-amino acid preprotein consisting of a signal peptide, a mature peptide, and a prodomain. Predicted mature Of-Pis1 was assumed to be a membrane-active AMP, based on the prediction of an amphipathic α-helical conformation with a net charge of +4. In addition, Of-Pis1 demonstrated significant similarities with other piscidin family members in terms of gene structure, sequence homology, and evolutionary relationship. Examination by quantitative real-time PCR (qPCR) of basal transcription of Of-Pis1 in the tissues of naïve rock bream, revealed predominant transcript levels in the gills, followed by the spleen, intestine, skin, and head kidney. In gill tissues, the temporally induced mRNA expression of Of-Pis1, upon in vivo injection trials with lipopolysaccharide (LPS); polyinosinic:polycytidylic acid (poly I:C); and pathogens, including Edwardsiella tarda, Streptococcus iniae, and rock bream iridovirus (RBIV), was weak. In contrast, in vivo flagellin administration led to a robust upregulation of Of-Pis1 in different tissues. Antimicrobial potency was determined by employing recombinant (rOf-Pis1), and synthetic (pOf-Pis1) peptides, in in vitro assays. Recombinant overexpression inhibited the growth of bacteria expressing the rOf-Pis1 protein in a growth delay assay. The broad antimicrobial spectrum of pOf-Pis1 was evidenced by its potent activity against an array of microbes, including bacteria, fungi, and parasitic species. In addition, pOf-Pis1 showed no significant hemolytic toxicity against human erythrocytes. Collectively, the data presented in the current study improve our understanding of the piscidin AMP family, and the contribution of Of-Pis1 to the rock bream immunity.

Chionodracine (Cnd) is a 22-residue peptide of the piscidin family expressed in the gills of the Chionodraco hamatus as protection from bacterial infections. Here, we report the effects of synthetic Cnd on both Psychrobacter sp. TAD1 and Escherichia coli bacteria, as well as membrane models. We found that Cnd perforates the inner and outer membranes of Psychrobacter sp. TAD1, making discrete pores that cause the cellular content to leak out. Membrane disruption studies using intrinsic and extrinsic fluorescence spectroscopy revealed that Cnd behaves similarly to other piscidins, with comparable membrane partition coefficients. Membrane accessibility assays and structural studies using NMR in detergent micelles show that Cnd adopts a canonical topology of antimicrobial helical peptides, with the hydrophobic face toward the lipid environment and the hydrophilic face toward the bulk solvent. The analysis of Cnd free energy of binding to vesicles with different lipid contents indicates a preference for charged phospholipids and a more marked binding to native E. coli extracts. Taken with previous studies on piscidin-like peptides, we conclude that Cnd first adsorbs to the membrane, and then forms pores together with membrane fragmentation. Since Cnd has only marginal hemolytic activity, it constitutes a good template for developing new antimicrobial agents.