Antimicrobial Peptides: Identification of two Beta-Defensins in a Teleost Fish, the European Sea Bass (Dicentrarchus labrax)

Abstract

Beta-defensins consist in a group of cysteine-rich antimicrobial peptides (AMPs), widely found throughout vertebrate species, including teleost fish, with antimicrobial and immunomodulatory activities. However, although the European sea bass (Dicentrarchus labrax) is one of the most commercially important farmed fish species in the Mediterranean area, the characterization of its beta-defensins and its potential applications are still missing. In this study, we characterized two members of the beta-defensin family in this species. Phylogenetic and synteny analysis places sea bass peptides in the beta-defensin subfamilies 1 and 2, sharing similar features with the other members, including the six cysteines and the tertiary structure, that consists in three antiparallel beta-sheets, with beta-defensin 1 presenting an extra alpha-helix at the N-terminal. Further studies are necessary to uncover the functions of sea bass beta-defensins, particularly their antimicrobial and immunomodulatory properties, in order to develop novel prophylactic or therapeutic compounds to be used in aquaculture production.

Keywords: European sea bass (Dicentrarchus labrax); antimicrobial peptides; aquaculture; beta-defensins.

Figure 1

Sea bass beta-defensin coding DNA and amino acid sequences. Nucleotides are indicated in the upper row, and amino acids are indicated in italic in the lower row. Mature peptides are boxed, and intron positions are indicated by arrows.

Figure 2

Alignment of sea bass beta-defensins. Cysteines are shaded gray, and the predicted disulphide bridges are also shown. Identical residues are denoted by (*), conserved substitutions by (:) and semi-conserved substitutions by (.).

Figure 3

Three dimensional models of sea bass beta-defensins 1 and 2. Conserved cysteines are highlighted. Beta-defensin 1 shows an additional alpha-helix at the N-terminal. Models were predicted using as templates human beta-defensin 6 (2lwl.1.A) for beta-defensin 1, and human beta-defensin 4 (5ki9.1.A) and oyster big defensin (6qbk.1.A) for beta-defensin 2.

Figure 4

Genomic organization of sea bass beta-defensin genes. (A) Exon/intron diagram of sea bass beta-defensin genes. (B) Comparative view with other vertebrate beta-defensins. Exons are shown as boxes and introns as solid lines, with sizes of exons/introns in base pairs.

Figure 5

Alignment of sea bass beta-defensins with peptides from other vertebrate species. Cysteines are shaded gray and are denoted by (*) and semi-conserved substitutions by (.).

Figure 6

Phylogenetic analysis of beta-defensin peptides. The evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model [32]. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed [33]. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [33]. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model, and then selecting the topology with superior log likelihood value. This analysis involved 56 amino acid sequences. All positions containing gaps and missing data were eliminated (complete deletion option). There was a total of 37 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [34].

Figure 7

Analysis of beta-defensin loci in the European sea bass (D. labrax, location HG916831.1), Barramundi perch (L. calcarifer, location CYIF01000095.1), Gilthead seabream (S. aurata, location LR537133.1; 13), Yellowtail amberjack (S. lalandi, locations PEQF01098735.1 and PEQF01099151.1 for beta-defensins 1 and 2, respectively) and Turbot (S. maximus, location CM023546.1; 4). Analysis was performed using sequences available in the Ensembl genome browser 103, and the corresponding accession numbers of each gene are described in Table S2. S. lalandi xrra1: incomplete gene.

Figure 8

Basal expression of beta-defensin genes in different organs of healthy sea bass, measured by real-time PCR. Each sample was normalized to beta actin (actb) calculated by the comparative CT method (2^−ΔΔCT). Values are presented as means ± standard deviation (S.D.) (n = 5).