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)

BackgroundBig defensin is an antimicrobial peptide composed of a highly hydrophobic N-terminal region and a cationic C-terminal region containing six cysteine residues involved in three internal disulfide bridges. While big defensin sequences have been reported in various mollusk species, few studies have been devoted to their sequence diversity, gene organization and their expression in response to microbial infections.FindingsUsing the high-throughput Digital Gene Expression approach, we have identified in Crassostrea gigas oysters several sequences coding for big defensins induced in response to a Vibrio infection. We showed that the oyster big defensin family is composed of three members (named Cg-BigDef1, Cg-BigDef2 and Cg-BigDef3) that are encoded by distinct genomic sequences. All Cg-BigDefs contain a hydrophobic N-terminal domain and a cationic C-terminal domain that resembles vertebrate β-defensins. Both domains are encoded by separate exons. We found that big defensins form a group predominantly present in mollusks and closer to vertebrate defensins than to invertebrate and fungi CSαβ-containing defensins. Moreover, we showed that Cg-BigDefs are expressed in oyster hemocytes only and follow different patterns of gene expression. While Cg-BigDef3 is non-regulated, both Cg-BigDef1 and Cg-BigDef2 transcripts are strongly induced in response to bacterial challenge. Induction was dependent on pathogen associated molecular patterns but not damage-dependent. The inducibility of Cg-BigDef1 was confirmed by HPLC and mass spectrometry, since ions with a molecular mass compatible with mature Cg-BigDef1 (10.7 kDa) were present in immune-challenged oysters only. From our biochemical data, native Cg-BigDef1 would result from the elimination of a prepropeptide sequence and the cyclization of the resulting N-terminal glutamine residue into a pyroglutamic acid.ConclusionsWe provide here the first report showing that big defensins form a family of antimicrobial peptides diverse not only in terms of sequences but also in terms of genomic organization and regulation of gene expression.

The cultivated Pacific oyster Crassostrea gigas has suffered for decades large scale summer mortality phenomenon resulting from the interaction between the environment parameters, the oyster physiological and/or genetic status and the presence of pathogenic microorganisms including Vibrio species. To obtain a general picture of the molecular mechanisms implicated in C. gigas immune responsiveness to circumvent Vibrio infections, we have developed the first deep sequencing study of the transcriptome of hemocytes, the immunocompetent cells. Using Digital Gene Expression (DGE), we generated a transcript catalog of up-regulated genes from oysters surviving infection with virulent Vibrio strains (Vibrio splendidus LGP32 and V. aestuarianus LPi 02/41) compared to an avirulent one, V. tasmaniensis LMG 20012T. For that an original experimental infection protocol was developed in which only animals that were able to survive infections were considered for the DGE approach. We report the identification of cellular and immune functions that characterize the oyster capability to survive pathogenic Vibrio infections. Functional annotations highlight genes related to signal transduction of immune response, cell adhesion and communication as well as cellular processes and defence mechanisms of phagocytosis, actin cytosqueleton reorganization, cell trafficking and autophagy, but also antioxidant and anti-apoptotic reactions. In addition, quantitative PCR analysis reveals the first identification of pathogen-specific signatures in oyster gene regulation, which opens the way for in depth molecular studies of oyster-pathogen interaction and pathogenesis. This work is a prerequisite for the identification of those physiological traits controlling oyster capacity to survive a Vibrio infection and, subsequently, for a better understanding of the phenomenon of summer mortality.

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.

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Protozoan parasites of the genus Perkinsus are considered important pathogens responsible for mass mortalities in several mollusk species worldwide. In the present study we describe for the first time a parasite of the genus Perkinsus infecting the mangrove oyster Crassostrea rhizophorae from the Brazilian coast. Prevalence of this parasite was low in the Pacoti River estuary (Ceará, northeast Brazil) and absent in oysters from southern Brazil. Oyster gill and rectum tissues incubated in Ray's fluid thioglycollate medium (RFTM) revealed the presence of spherical hypnospores (5 to 55 µm diam.). Histological analysis showed the occurrence of typical signet-ring trophozoites and schizonts (3 to 6 µm diam.) infecting connective tissues of several organs and digestive epithelia. PCR assays specific to the genus Perkinsus, followed by cloning and sequencing of the internal transcribed spacer (ITS) region of the ribosomal ribonucleic acid (rRNA) gene complex, confirmed a close phylogenetic relationship between Brazilian Perkinsus sp. and P. beihaiensis infecting Chinese oysters. KEY WORDS: Crassostrea rhizophorae · Mangrove oyster · Perkinsus sp. · Protozoan parasite · PCR-RFLP · rRNA · RFTM · Sporulation Resale or republication not permitted without written consent of the publisherDis Aquat Org 88: [13][14][15][16][17][18][19][20][21][22][23] 2009 eases affecting edible and cultured bivalve species from 2 Brazilian regions. The project was designed to survey parasites among wild and cultivated populations of oysters Crassostrea rhizophorae and C. gigas from Santa Catarina Island and the Pacoti River estuary (Fortaleza, in the state of Ceará, northeast Brazil). The results provide data on distributions of pathogens and on host susceptibilities to pathogens, which will qualify further monitoring programs, help avoid disease transfers from infected populations, and inform efforts to keep unaffected areas parasite-free.The first described parasite of the genus Perkinsus was P. marinus (Mackin et al. 1950), which was identified as being responsible for mortality outbreaks among Crassostrea virginica oyster stocks from Louisiana, USA. It was first described as Dermocystidium marinum, based partly on characteristics of hypertrophic hypnospores and zoosporangia that enlarge among infected oyster tissues when they are incubated in a high-salt formulation of Ray's fluid thioglycollate medium (RFTM) (Ray 1952). Since that time, diverse Perkinsus spp. have been reported to infect many important commercially cultured mollusk species worldwide.Perkinsus olseni was first described as a pathogen of Australian abalone Haliotis ruber (Lester & Davis 1981). Azevedo (1989) described the species P. atlanticus infecting the carpet shell clam Ruditapes decussatus in Portugal. P. olseni, which is currently synonymous with P. atlanticus, has wide geographic and host ranges. It has subsequently been reported in other bivalve species, such as Pitar rostrata from Uruguay (Cremonte et al. 2005) and R. philippinarum, Venerupis pullast...