Characterization of a Defensin from the Oyster Crassostrea gigas

Abstract

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 Grampositive bacteria but showed no or limited activities against Gramnegative 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 ␣-␤ motif (CS-␣␤) 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 Cgdef 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. Antimicrobial peptides (AMPs)5 are important components of the innate immune system that have been conserved during evolution (1). They constitute a first line of host defense against pathogens in plants and animals (2, 3). We estimate that more than 1000 antimicrobial peptides have been described at the level of their primary structure (2). They are gathered in the Antimicrobial Sequence Database (www.bbcm.units.it/ϳtossi/amsdb.html). AMPs can be classified into three major groups: (i) linear peptides that form amphipathic ␣-helices, (ii) cyclic peptides containing cysteine-residue engaged in disulfide bonds, and (iii) peptides with an overrepresentation in certain amino acids (proline, arginine, glycine, or histidine). Despite their great diversity in terms of size, primary structure, amino acid composition, and mode of action, most AMPs are characterized by the preponderance in cationic and hydrophobic amino acids (2). In most of the cases, this amphipathic character is considered as crucial for the interaction of the effective peptide with the membrane of sensitive microorganisms. This first interaction seems to be essential whatever the exact mode of action: (i) through disruption of their negatively charged cytoplasmic membranes or (ii) through killing following translocation into the bacteria without membrane lyses and binding to a specific target pr...

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

“…In Drosophila melanogaster, the expression of genes encoding antimicrobial peptides is induced in the epithelia of the respiratory tract, the oral region, the digestive tract, the malpighian tubules, and the male and female reproductive tracts (37). Interestingly, a defensin named Cg-Def has been characterized from the mantle of the oyster C. gigas (38) where it may colocalize with Cg-BPI. The oyster, which is continuously exposed to an environment rich in microorganisms, may have developed an efficient system to limit bacterial invasion.…”

Insight into Invertebrate Defensin Mechanism of Action

“…and Cg-Defh2 peptides were obtained according to the method described for recombinant Cg-Defm production with minor modifications (7). Recombinant Cg-Defh1 and Cg-Defh2 were expressed in Escherichia coli Rosetta (DE3) as an N-terminal His 6 -tagged fusion protein using the pET-28a system (Novagen).…”

“…Expression of Cg-Defs was performed as described previously (7). Modifications in purification procedure were introduced to improve the production yield.…”

“…The 80% acetonitrile fraction containing the peptide mixture was then frozen and lyophilized. The methionine residue introduced at the peptide N terminus was subjected to CNBr cleavage as described previously (7).…”

“…First evidenced in scorpion toxins, this motif is widespread in invertebrate defensins like arthropod and mollusk defensins (5-7), but it is also found in plectasin, a defensin from the saprophytic ascomycete Pseudoplectania nigrella (8). CS␣␤-containing defensins are mostly active against Gram-positive bacteria (7)(8)(9). Conversely, mammalian ␣-and ␤-defensins are usually both active against Grampositive and -negative bacteria (10).…”