Julie Fievet

Characterization of a Defensin from the Oyster Crassostrea gigas RECOMBINANT PRODUCTION, FOLDING, SOLUTION STRUCTURE, ANTIMICROBIAL ACTIVITIES, AND GENE EXPRESSION

In invertebrates, defensins were found in arthropods and in the mussels. Here, we report for the first time the identification and characterization of a defensin (Cg-Def) from an oyster. Cg-def mRNA was isolated from Crassostrea gigas mantle using an expressed sequence tag approach. To gain insight into potential roles of Cg-Def in oyster immunity, we produced the recombinant peptide in Escherichia coli, characterized its antimicrobial activities, determined its solution structure by NMR spectroscopy, and quantified its gene expression in vivo following bacterial challenge of oysters. Recombinant Cg-Def was active in vitro against Gram-positive bacteria but showed no or limited activities against Gram-negative bacteria and fungi. The activity of Cg-Def was retained in vitro at a salt concentration similar to that of seawater. The Cg-Def structure shares the so-called cystine-stabilized α-β motif (CS-αβ) with arthropod defensins but is characterized by the presence of an additional disulfide bond, as previously observed in the mussel defensin (MGD-1). Nevertheless, despite a similar global fold, the Cg-Def and MGD-1 structures mainly differ by the size of their loops and by the presence of two aspartic residues in Cg-Def. Distribution of Cg-def mRNA in various oyster tissues revealed that Cg-def is mainly expressed in mantle edge where it was detected by mass spectrometry analyses. Furthermore, we observed that the Cg-def messenger concentration was unchanged after bacterial challenge. Our results suggest that Cg-def gene is continuously expressed in the mantle and would play a key role in oyster by providing a first line of defense against pathogen colonization.

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.

Big Defensins, a Diverse Family of Antimicrobial Peptides That Follows Different Patterns of Expression in Hemocytes of the Oyster Crassostrea gigas

Background Big 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. Findings Using 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. Conclusions We 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.

A new class (penaeidin class 4) of antimicrobial peptides from the Atlantic white shrimp (Litopenaeus setiferus) exhibits target specificity and an independent proline-rich-domain function

A highly pure, chemically defined representative of a new class of antimicrobial peptide from the Atlantic white shrimp (Litopenaeus setiferus), penaeidin class 4 [Pen4-1 (penaeidin class 4 isoform 1)], was produced synthetically. Chemical synthesis was achieved by native ligation from two separate domains yielding a bioactive peptide that reflected the characteristics of native penaeidin. Synthetic Pen4-1 proved to be an effective antimicrobial peptide, particularly against the broad-spectrum pathogen Fusarium oxysporum, exhibiting a complex effect on reproductive growth at inhibitory concentrations resulting in the suppression of spore formation. Pen4-1 exhibits unique features [not previously observed for penaeidins from the Pacific white shrimp (L. vannamei)], including target-species specificity against Gram-positive bacteria, indicating a potential partitioning of antimicrobial function among this family of peptides. The proline-rich domain of penaeidin class 4 alone was an active antimicrobial peptide, having the same target range as the full-length Pen4-1. These findings indicate that the proline-rich domain of penaeidin is sufficient to confer target specificity and that divergence in this domain between classes can result in a gain in antimicrobial function as observed for the proline-rich domain of Pen4-1.

A relationship between antimicrobial peptide gene expression and capacity of a selected shrimp line to survive a Vibrio infection

Understanding of antimicrobial defence mechanisms of penaeid shrimp should help in the design of efficient strategies for the management and disease control in aquaculture. In this study, we have specifically analysed the expression in circulating hemocytes of antimicrobial peptides (AMPs) encoding genes, such as PEN2 and PEN3, ALF, crustin, lysozyme and a putative cysteine-rich peptide. We evidenced a relationship between the level of expression of some AMPs and the successful response of the shrimp, Litopenaeus stylirostris, to circumvent a pathogenic Vibrio penaeicida infection. Additionally, significant differences in some AMP transcript amounts are evidenced between control, non-selected shrimp line and the third generation breeding of shrimp selected for their survival to natural V. penaeicida infections. On the basis of these results, it will now be of great interest to determine if these AMPs are directly involved in the resistance of shrimp to infection or if they only reflect other acquired defence mechanisms which can confer a resistance.

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.

Influence of water temperature and food on the last stages of cultured pearl mineralization from the black-lip pearl oyster Pinctada margaritifera

Environmental parameters, such as food level and water temperature, have been shown to be major factors influencing pearl oyster shell growth and molecular mechanisms involved in this biomineralization process. The present study investigates the effect of food level (i.e., microalgal concentration) and water temperature, in laboratory controlled conditions, on the last stages of pearl mineralization in order to assess their impact on pearl quality. To this end, grafted pearl oysters were fed at different levels of food and subjected to different water temperatures one month prior to harvest to evaluate the effect of these factors on 1) pearl and shell deposition rate, 2) expression of genes involved in biomineralization in pearl sacs, 3) nacre ultrastructure (tablet thickness and number of tablets deposited per day) and 4) pearl quality traits. Our results revealed that high water temperature stimulates both shell and pearl deposition rates. However, low water temperature led to thinner nacre tablets, a lower number of tablets deposited per day and impacted pearl quality with better luster and fewer defects. Conversely, the two tested food level had no significant effects on shell and pearl growth, pearl nacre ultrastructure or pearl quality. However, one gene, Aspein, was significantly downregulated in high food levels. These results will be helpful for the pearl industry. A wise strategy to increase pearl quality would be to rear pearl oysters at a high water temperature to increase pearl growth and consequently pearl size; and to harvest pearls after a period of low water temperature to enhance luster and to reduce the number of defects.