Bernard Romestand

Insights into the anti-microbial defense of marine invertebrates: the penaeid shrimps and the oyster Crassostrea gigas

Summary:  Research on innate immunity of the penaeid shrimps and the oyster Crassostrea gigas is motivated greatly by economical necessities. Indeed, the aquaculture of these organisms is now limited by the development of infectious diseases. Studying anti‐microbial peptides/proteins (AMPs), which are effector molecules of the host defense, is particularly attractive not only for progressing basic knowledge on immunity but also because they offer various possible applications for disease management in aquaculture. AMPs are explored with a global approach, considering their structure, properties, function, gene expression, and tissue distribution during the response to infections. In shrimp, investigations of the penaeidins, which are constitutively expressed peptides, have highlighted the importance of hemocytes and hematopoiesis as major elements of the immune response, providing both local and systemic reactions. The activation of hematopoiesis must be regarded as a regulatory way for the expression and distribution of constitutively expressed immune effectors. As complementary approaches, genomics and gene profiling are promising to deepen our understanding of the anti‐microbial defense of the oyster and the shrimp. However, real progress will depend also on the characterization of hemocyte lineages and hematopoiesis of these marine invertebrates as well as on the ontogenesis of their immune systems.

In vitro production of superoxide and nitric oxide (as nitrite and nitrate) by Mytilus galloprovincialis haemocytes upon incubation with PMA or laminarin or during yeast phagocytosis

The phagocytic process is one of the most important elements of the self-defence system in mammals as well as in molluscs. In mammalian phagocytes, superoxide participates in the innate defence system by combining with nitric oxide to generate peroxynitrite, a strong oxidant that possesses highly cytotoxic properties against bacteria. To evidence a role of nitric oxide in the self-defence system of the marine bivalve Mytilus galloprovincialis similar to the role observed in the mammalian defence system, we measured the generation of superoxide and nitrite/nitrate (the stable end products of nitric oxide) upon in vitro stimulation of M. galloprovincialis haemocytes with PMA, laminarin, LPS and by phagocytosis of Saccharomyces cerevisiae (yeast cells). We show that stimulation with PMA, laminarin and yeast cell phagocytosis promotes superoxide and nitrite/nitrate generation from M. galloprovincialis haemocytes. Inhibitors of NADPH oxidase and inhibitors of NO synthase decreased the nitrite/nitrate levels generated by M. galloprovincialis haemocytes showing that both NADPH oxidase and NO synthase pathways are involved in the self-defence system of M. galloprovincialis.

Evidence of a bactericidal permeability increasing protein in an invertebrate, the Crassostrea gigas Cg-BPI

A cDNA sequence with homologies to members of the LPS-binding protein and bactericidal/permeability-increasing protein (BPI) family was identified in the oyster Crassostrea gigas. The recombinant protein was found to bind LPS, to display bactericidal activity against Escherichia coli, and to increase the permeability of the bacterial cytoplasmic membrane. This indicated that it is a BPI rather than an LPS-binding protein. By in situ hybridization, the expression of the C. gigas BPI (Cg-bpi) was found to be induced in hemocytes after oyster bacterial challenge and to be constitutive in various epithelia of unchallenged oysters. Thus, Cg-bpi transcripts were detected in the epithelial cells of tissues/organs in contact with the external environment (mantle, gills, digestive tract, digestive gland diverticula, and gonad follicles). Therefore, Cg-BPI, whose expression profile and biological properties are reminiscent of mammalian BPIs, may provide a first line of defense against potential bacterial invasion. To our knowledge, this is the first characterization of a BPI in an invertebrate.

Use of OmpU porins for attachment and invasion of Crassostrea gigas immune cells by the oyster pathogen Vibrio splendidus.

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 Cg-EcSOD, 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.

Philasterides dicentrarchi, n. sp., (ciliophora, scuticociliatida), a histophagous opportunistic parasite of Dicentrarchus labrax (Linnaeus, 1758), a reared marine fish

Summary In sea bass Dicentrarchus labrax (L.) reared in the Mediterranean sea, mortality suddenly arose. The diseased fishes, noticeable when almost dead, were massively infested with a histophagous ciliate. Depending on the infected host organs, the ciliate showed in its food vacuoles various categories of ingested cells: erythrocytes, ovocytes, and others. The infecting mode of this presumed “parasite” is still unknown, all experimental infections having failed. Morphological, biometric and ultrastructural studies lead to the conclusion that, because of its morphological characteristics and its main stomatogenesis processes, this ciliate is a Scuticociliatida Philasterina. Compared to other known species, it differs by its small size, the small and regular number of its somatic kineties, the position of its buccal organelles (and, mainly, the bipartite paroral membrane), the presence of two types of extrusomes, and a perinuclear envelope. All these characteristics justify the creation of a new species: Philasterides dicentrarchi. This particular ciliate habitat is compared to that of other commensal or opportunistic parasitic ciliates of invertebrates and marine fishes.

The major outer membrane protein OmpU of Vibrio splendidus contributes to host antimicrobial peptide resistance and is required for virulence in the oyster Crassostrea gigas

Vibrio splendidus, strain LGP32, is an oyster pathogen associated with the summer mortalities affecting the production of Crassostrea gigas oysters worldwide. Vibrio splendidus LGP32 was shown to resist to up to 10 microM Cg-Def defensin and Cg-BPI bactericidal permeability increasing protein, two antimicrobial peptides/proteins (AMPs) involved in C. gigas immunity. The resistance to both oyster Cg-Def and Cg-BPI and standard AMPs (polymyxin B, protegrin, human BPI) was dependent on the ompU gene. Indeed, upon ompU inactivation, minimal bactericidal concentrations decreased by up to fourfold. AMP resistance was restored upon ectopic expression of ompU. The susceptibility of bacterial membranes to AMP-induced damages was independent of the ompU-mediated AMP resistance. Besides its role in AMP resistance, ompU proved to be essential for the adherence of V. splendidus LGP32 to fibronectin. Interestingly, in vivo, ompU was identified as a major determinant of V. splendidus pathogenicity in oyster experimental infections. Indeed, the V. splendidus-induced oyster mortalities dropped from 56% to 11% upon ompU mutation (Kaplan-Meier survival curves, P < 0.01). Moreover, in co-infection assays, the ompU mutant was out competed by the wild-type strain with competitive indexes in the range of 0.1-0.2. From this study, ompU is required for virulence of V. splendidus. Contributing to AMP resistance, conferring adhesive properties to V. splendidus, and being essential for in vivo fitness, the OmpU porin appears as an essential effector of the C. gigas/V. splendidus interaction.

Stylicins, a new family of antimicrobial peptides from the Pacific blue shrimp Litopenaeus stylirostris

The present study reports the characterization of Ls-Stylicin1, a novel antimicrobial peptide from the penaeid shrimp, Litopenaeus stylirostris. The predicted mature peptide of 82 residues is negatively charged (theoretical pI=5.0) and characterized by a proline-rich N-terminal region and a C-terminal region containing 13 cysteine residues. The recombinant Ls-Stylicin1 has been isolated in both monomeric and dimeric forms. Both display strong antifungal activity against Fusarium oxysporum (1.25 microM<MIC<2.5 microM), a pathogenic fungus of shrimp, but lower antimicrobial activity against Gram (-) bacteria, Vibrio sp. (40 microM<MIC<80 microM). However, rLs-Stylicin1 is able to agglutinate Vibrio penaeicidae in vitro in agreement with its potent LPS-binding activity on immobilized LPS of V. penaeicidae (dissociation constant (K(d)) of 9.6x10(-8)M). This molecule with no evident homology to other hitherto described antimicrobial peptides but identified herein several species of penaeid shrimp is thought to be the first member of a shrimp antimicrobial peptide family, which we termed stylicins.

Functional Divergence in Shrimp Anti- Lipopolysaccharide Factors (ALFs): From Recognition of Cell Wall Components to Antimicrobial Activity

Antilipopolysaccharide factors (ALFs) have been described as highly cationic polypeptides with a broad spectrum of potent antimicrobial activities. In addition, ALFs have been shown to recognize LPS, a major component of the Gram-negative bacteria cell wall, through conserved amino acid residues exposed in the four-stranded β-sheet of their three dimensional structure. In penaeid shrimp, ALFs form a diverse family of antimicrobial peptides composed by three main variants, classified as ALF Groups A to C. Here, we identified a novel group of ALFs in shrimp (Group D ALFs), which corresponds to anionic polypeptides in which many residues of the LPS binding site are lacking. Both Group B (cationic) and Group D (anionic) shrimp ALFs were produced in a heterologous expression system. Group D ALFs were found to have impaired LPS-binding activities and only limited antimicrobial activity compared to Group B ALFs. Interestingly, all four ALF groups were shown to be simultaneously expressed in an individual shrimp and to follow different patterns of gene expression in response to a microbial infection. Group B was by far the more expressed of the ALF genes. From our results, nucleotide sequence variations in shrimp ALFs result in functional divergence, with significant differences in LPS-binding and antimicrobial activities. To our knowledge, this is the first functional characterization of the sequence diversity found in the ALF family.

Dendrigraft Poly-L-lysine: A Non-Immunogenic Synthetic Carrier for Antibody Production

An easily synthesized DendriGraft poly-lysine DGL-G3 (third generation) was shown to act as an efficient carrier for raising antibodies directed against small molecules. The immunological properties of three different forms of DGL-G3 were investigated: the native form (molecular weight 22 kDa bearing a mean number of 123 surface amino groups as TFA salts), a form modified at the C-terminus by fluorescein (fluorescein-DGL-G3), and last a surface-modified form bearing histamine (DGL-G3-Histamine). Our studies demonstrate the native DGL-G3 to be inefficient in eliciting antibody production in rabbits. Immunizations of rabbits using the core-modified fluorescein-DGL-G3 or the surface-modified DGL-G3-histamine conjugate failed in eliciting antibody production. Conversely, following a primary immunization using a BSA-histamine conjugate, a second immunization with DGL-G3-histamine conjugate improved the production of specific hapten-directed antibodies, which demonstrates the utility of DGL-G3 as a carrier for the production of highly specific antibody against haptens.

Nitrite released in haemocytes from Mytilus galloprovincialis, Crassostrea gigas and Ruditapes decussatus upon stimulation with phorbol myristate acetate

In order to demonstrate the involvement of nitric oxide in the defence systems of marine bivalves, we investigated the production of superoxide and nitrite, following in vitro phorbol myristate acetate stimulation of Mytilus galloprovincialis, Crassostrea gigas and Ruditapes decussatus haemocytes. Whereas M. galloprovincialis and C. gigas haemocytes were found to produce superoxide and nitrite, R. decussatus haemocytes were found to be unable to generate either of these mediators. Nitrite is a stable end product of nitric oxide and peroxynitrite as well; it appeared therefore that some marine bivalves, to kill microbial pathogens, use NADPH-oxidase and nitric oxide-synthase pathways. This was confirmed at an experimental level where inhibitors of both enzymatic pathways blocked the production of nitrite. Moreover, this notion was strengthened by the inability of the haemocytes from R. decussatus, which cannot produce superoxide, to release nitrite when stimulated.