Molecular diversity of antimicrobial effectors in the oyster Crassostrea gigas

Abstract

Background: To gain insight into the molecular diversity of antimicrobial peptides and proteins in the oyster Crassostrea gigas, we characterized and compared the sequence polymorphism of the antimicrobial peptides (AMPs), Cg-Defensins (Cg-Defs) and Cg-Proline Rich peptide (Cg-Prp), and of the bactericidal permeability increasing protein, Cg-BPI. For that, we analyzed genomic and transcript sequences obtained by specific PCR amplification and in silico searches.

Results: High diversification among the three antimicrobial effectors was evidenced by this polymorphism survey. On the basis of sequence phylogenies, each AMP aggregates into clearly defined groups of variants and is the product of a multigenic family displaying a variety of gene structures. In contrast, Cg-bpi forms a single group and is encoded by a single gene copy. Moreover, we identified for both AMPs several genetic mechanisms of diversification such as recombination, parallel mutations leading to phylogenetic homoplasy and indel events. In addition, the non synonymous to synonymous substitutions ratio by codon (dN/dS) revealed several negatively and positively selected sites for both AMPs, suggesting that directional selection pressures have shaped their sequence variations.

Conclusions: This study shows for the first time in a mollusc that antimicrobial peptides and proteins have been subject to distinct patterns of diversification and we evidence the existence of different evolutionary routes leading to such sequence variability.

Figure 1

Phylogenies from CDS of three antimicrobial effectors and non immune gene from C. gigas. a. Cg-defs: I. Cg-defh1 (black triangles); II. Cg-defh2 (white triangles) and III. Cg-defm (dark gray triangles). Recombinant sequences between Cg-defh1 and Cg-defh2 are indicated (light gray triangles). b. Cg-prp: I. Cg-prp short forms (black circles), II. Cg-prp short and long forms, and III. Cg-prp long forms (white circles). Cg-prp recombinant sequences are indicated (gray circles). c. Cg-bpi (white rhombus) and d. Cg-actin (white squares). Construction of cladograms were performed with PHYML v2.4.4 using the models GTR+Γ for Cg-defs, HKY85+Γ for Cg-prp, TN93+I for Cg-bpi and F84+Γ for Cg-actin determined by the Akaike information criterion. For accurate comparison between the four genes, cladograms were drawn in the same scale.

Figure 2

Gene organization of Cg-defs and Cg-prp from C. gigas. a. Cg-prp genomic structures. Black boxes indicate signal peptide region; white boxes indicate anionic domain region and striped boxes indicate cationic domain region. b. Cg-defhs and Cg-defm gene structures. Black boxes indicate signal peptide region and white boxes indicate mature peptide region. Numbers indicate the length of exons and introns (bases pairs). Note the gene size is not to scale.

Figure 3

Cladograms Cg-defs and Cg-prp and schematic representation of aligned CDS with synonymous and nonsynonymous substitutions. a. Cg-defs. b. Cg-prp. Identical sequences are grouped and the number is indicated at the right of each branch of cladograms and sequence symbols are the same as in figure 1. Nonsynonymous (blue boxes) and synonymous substitutions (yellow boxes) are localized over schematic alignment. Different groups of sequences (I, II and III) and obvious recombinants (R) are boxed. Indel events are represented by dark lines (Cg-prp) and striped box (Cg-defs) and circles indicate parallel homoplasic mutations. SP: signal peptide; MP: Mature peptide; AD: Anionic Domain; CD: Cationic Domain. For accurate comparison between AMPs, cladograms were drawn in the same scale.

Figure 4

Alignments of amino acids sequences of putative isoforms for Cg-defs and Cg-prp from C. gigas. a. Cg-Defm (FJ669323-FJ669336) and Cg-defhs (FJ669341- FJ669352). b. Cg-Prp (FJ669354- FJ669402). Highlighted sites showed evidence of positive (in black) or negative (in gray) selection (p < 0.05). Dots indicate identical residues and gaps (-) were introduced to obtain maximum sequence identity. The different domains are indicated in white boxes at the bottom of alignments.