Cg-Prp

Diverse families of antimicrobial peptides and proteins have been described in oysters. We investigated here how antimicrobials are involved in the immune response against a pathogenic strain of Vibrio splendidus. Oyster antimicrobials were shown to display a wide variety of expression profiles in hemocyte populations and tissues. Oyster defensins are constitutively expressed in specific tissues such as mantle (Cg-Defm) or hemocytes (Cg-Defhs), while Cg-BPI is inducible and Cg-Prp appears down-regulated in hemocytes upon infection. The migratory behavior of hemocytes that express the different antimicrobials was found to be involved in the oyster response to a pathogenic Vibrio infection. Indeed, it contributes to colocalize several antimicrobials that were shown here to have synergistic activities. We propose that such a synergy, which was evidenced both within and between families of antimicrobials, might compensate for the low concentration of antimicrobials in oyster tissues.

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.

A cDNA sequence that encodes a 61-amino acid polypeptide precursor with homologies to proline-rich antimicrobial peptides (AMPs) was identified in the oyster Crassostrea gigas. After release of a hydrophobic signal peptide, the resulting 37-amino acid peptide, Cg-Prp, is composed of an acidic region and a cationic proline-rich region. To evaluate the biological properties of Cg-Prp, multiple proline-rich peptides corresponding to putative processing of the full-length Cg-Prp were synthesized. A limited antimicrobial activity was observed for two of them, which also showed strong synergistic antimicrobial activity with Cg-Def, a defensin from C. gigas. To our knowledge, this is the first evidence of synergy between a defensin and another AMP in an invertebrate. By in situ hybridization, the expression of Cg-prp was found to be restricted to hemocytes and induced following bacterial challenge. Cg-prp transcripts were also detected in hemocytes infiltrating mantle, where Cg-Def is expressed. Additionally, by immunocytochemistry, we showed that Cg-Prp or one of its variants is present in some hemocytes together with defensins. In conclusion, we described here the first proline-rich AMP from mollusk. From our study, it is likely to provide a first line of defense against bacterial invasion by acting through synergy with defensins.