Molecular diversity of antimicrobial effectors in the oyster Crassostrea gigas

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. Background The Pacific oyster, Crassostrea gigas, is a filter feeder bivalve mollusc which is continually exposed to microorganisms naturally present in the marine environment. Thus, the capacity to control the microflora and overcome infections is essential for the oyster survival. The hemocytes, immunocompetent cells, play a central role in innate antimicrobial response. Indeed, in molluscs, hemocytes infiltrate injured tissues, phagocyte microorganisms, produce antimicrobial peptides (AMPs) and release factors such as lectins and reactive oxygen species (ROS) [1-3]. In C. gigas, several antimicrobial effectors have been recently characterized. An AMP, member of the defensin family named Cg-Defm, was identified from the oyster mantle [4]. Then, two additional defensins named Cg-Defhs (Cg-Defh1 and Cg-Defh2)have been characterized from hemocytes [5]. Both Cg-defm and Cg-defhs appear to be continuously expressed in different tissues. More recently, a new peptide, Cg-Prp, which belongs to the proline-rich AMP family, has been found in hemocytes [6]. Whereas Cg-Prp displays alone a weak in vitro antimicrobial activity, it revealed synergistic activity with Cg-Defm against both Gram positive and negative bacteria [6]. Additionally, a member of the bactericidal/permeability-increasing protein (BPI) family has been characterized [7]. Cg-bpi expression is induced in hemocytes after oyster bacterial challenge and constitutive in various tissue epithelia of unchallenged oysters. Similar to vertebrate BPIs, the oyster BPI binds LPS, displays bactericidal activity against Gram negative bacteria and increases the permeability of the bacterial cytoplasmic membrane [7]. High levels of sequence diversity were reported to be a characteristic of several antimicrobial effectors pertaining to the innate immunity system of invertebrates [8]. * Correspondence: Julien.De. Lorgeril@ifremer.fr Ifremer, CNRS, Université de Montpellier II, IRD, UMR 5119 « Ecosystèmes Lagunaires », Place Eugène Bataillon, CC80, 34095 Montpellier Cedex 5 (France Schmitt et al. BMC Evolutionary Biology 2010, 10:23 http://www.biomedcentral.com/1471-2148/10/23 © 2010 Schmitt et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The functional significance of this diversity has been addressed in some cases. Multiple variants of lectins from the horseshoe crab Carcinoscorpius rotundicauda are able to recognize and differentiate bacteria and fungi [9]. The AMP drosomycin from Drosophila melanogaster presents six isoforms with different antifungal activities [10]. Thus, diversification of antimicrobial effectors by accumulation of multiple variations around an originally unique form may provide significant means of acquiring microbial target specificity [11], concerned in the evolutionary arms race between pathogens and their hosts. In this work, we have investigated the diversity of three oyster antimicrobial effectors and shown the existence of a high diversity in the transcript and genomic sequences of three antimicrobials, two peptides (Cg-prp and Cg-defs) and one protein (Cg-bpi). We also evidence distinct phylogenies between them and propose the existence of a combination of different genetic mechanisms from which sequence variability arises.