OmpU porins are increasingly recognized as key determinants of pathogenic host Vibrio interactions. Although mechanisms remain incompletely understood, various species, including the human pathogen Vibrio cholera, require OmpU for host colonization and virulence. We have shown previously that OmpU is essential for virulence in the oyster pathogen Vibrio splendidus LGP32. Here, we showed that V. splendidus LGP32 invades the oyster immune cells, the hemocytes, through subversion of host-cell actin cytoskeleton. In this process, OmpU serves as an adhesin/invasin required for β-integrin recognition and host cell invasion. Furthermore, the major protein of oyster plasma, the extracellular superoxide dismutase CgEcSOD, is used as an opsonin mediating the OmpU-promoted phagocytosis through its RGD sequence. Finally, the endocytosed bacteria were found to survive intracellularly, evading the host defense by preventing acidic vacuole formation and limiting reactive oxygen species production. We conclude that (i) V. splendidus is a facultative intracellular pathogen that manipulates host defense mechanisms to enter and survive in host immune cells, and (ii) that OmpU is a major determinant of host cell invasion in Vibrio species, used by V. splendidus LGP32 to attach and invade oyster hemocytes through opsonisation by the oyster plasma Cg-EcSOD.T he oyster pathogen, Vibrio splendidus strain LGP32 was isolated from massive mortality events in the production of Crassostrea gigas oysters (1). However, up to now, little has been known about the route of infection and pathogenic processes of LGP32 (2, 3). A metalloprotease has been associated with toxicity (4, 5) and the outer membrane protein (OMP) OmpU was shown to be a major determinant of LGP32 virulence (6).As bacterial surface components, OMPs are both used by hosts for pathogen recognition and by pathogens for interaction with and invasion of host cells, serving as adhesion proteins (adhesins)

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.

Healthy oysters are inhabited by abundant microbial communities that vary with environmental conditions and coexist with immunocompetent cells in the circulatory system. In Crassostrea gigas oysters, the antimicrobial response, which is believed to control pathogens and commensals, relies on potent oxygen-dependent reactions and on antimicrobial peptides/proteins (AMPs) produced at low concentrations by epithelial cells and/or circulating hemocytes. In non-diseased oysters, hemocytes express basal levels of defensins (Cg-Defs) and proline-rich peptides (Cg-Prps). When the bacterial load dramatically increases in oyster tissues, both AMP families are driven to sites of infection by major hemocyte movements, together with bactericidal permeability/increasing proteins (Cg-BPIs) and given forms of big defensins (Cg-BigDef), whose expression in hemocytes is induced by infection. Co-localization of AMPs at sites of infection could be determinant in limiting invasion as synergies take place between peptide families, a phenomenon which is potentiated by the considerable diversity of AMP sequences. Besides, diversity occurs at the level of oyster AMP mechanisms of action, which range from membrane lysis for Cg-BPI to inhibition of metabolic pathways for Cg-Defs. The combination of such different mechanisms of action may account for the synergistic activities observed and compensate for the low peptide concentrations in C. gigas cells and tissues. To overcome the oyster antimicrobial response, oyster pathogens have developed subtle mechanisms of resistance and evasion. Thus, some Vibrio strains pathogenic for oysters are equipped with AMP-sensing systems that trigger resistance. More generally, the known oyster pathogenic vibrios have evolved strategies to evade intracellular killing through phagocytosis and the associated oxidative burst.

One contribution of 13 to a theme issue 'Evolutionary ecology of arthropod antimicrobial peptides'. Aquaculture contributes more than one-third of the animal protein from marine sources worldwide. A significant proportion of aquaculture products are derived from marine protostomes that are commonly referred to as 'marine invertebrates'. Among them, penaeid shrimp (Ecdysozosoa, Arthropoda) and bivalve molluscs (Lophotrochozoa, Mollusca) are economically important. Mass rearing of arthropods and molluscs causes problems with pathogens in aquatic ecosystems that are exploited by humans. Remarkably, species of corals (Cnidaria) living in non-exploited ecosystems also suffer from devastating infectious diseases that display intriguing similarities with those affecting farmed animals. Infectious diseases affecting wild and farmed animals that are present in marine environments are predicted to increase in the future. This paper summarizes the role of the main pathogens and their interaction with host immunity, with a specific focus on antimicrobial peptides (AMPs) and pathogen resistance against AMPs. We provide a detailed review of penaeid shrimp AMPs and their role at the interface between the host and its resident/pathogenic microbiota. We also briefly describe the relevance of marine invertebrate AMPs in an applied context. This article is part of the themed issue 'Evolutionary ecology of arthropod antimicrobial peptides'.

Background: How antimicrobial histones participate in invertebrate defense was still unclear. Results: Upon injury or infection, oyster immune cells release antimicrobial histones and extracellular DNA traps in a ROSdependent manner. Conclusion: DNA traps are involved in the defense of Lophotrochozoa. Their mechanistic bases are shared with vertebrates. Significance: This is a novel mechanism in the evolutionary conserved invertebrate immune arsenal.

BackgroundTo 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.ResultsHigh 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.ConclusionsThis 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.

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.

Three oyster defensin variants (Cg-Defh1, Cg-Defh2, and CgDefm) were produced as recombinant peptides and characterized in terms of activities and mechanism of action. In agreement with their spectrum of activity almost specifically directed against Gram-positive bacteria, oyster defensins were shown here to be specific inhibitors of a bacterial biosynthesis pathway rather than mere membrane-active agents. Indeed, at lethal concentrations, the three defensins did not compromise Staphylococcus aureus membrane integrity but inhibited the cell wall biosynthesis as indicated by the accumulation of the UDP-Nacetylmuramyl-pentapeptide cell wall precursor. In addition, a combination of antagonization assays, thin layer chromatography, and surface plasmon resonance measurements showed that oyster defensins bind almost irreversibly to the lipid II peptidoglycan precursor, thereby inhibiting the cell wall biosynthesis. To our knowledge, this is the first detailed analysis of the mechanism of action of antibacterial defensins produced by invertebrates. Interestingly, the three defensins, which were chosen as representative of the oyster defensin molecular diversity, bound differentially to lipid II. This correlated with their differential antibacterial activities. From our experimental data and the analysis of oyster defensin sequence diversity, we propose that oyster defensin activity results from selective forces that have conserved residues involved in lipid II binding and diversified residues at the surface of oyster defensins that could improve electrostatic interactions with the bacterial membranes.