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 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 alpha-beta motif (CS-alphabeta) 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.
Highlights
Messenger concentration was unchanged after bacterial challenge
In oyster, antimicrobial activities have been detected in the hemolymph of some species, Antimicrobial peptides (AMPs) have never been fully characterized despite many attempts to purify, from hemolymph and other tissues, such molecules by using reverse-phase high performance liquid chromatography (RP-HPLC) approaches [13]
As already observed for the mussel defensins MGD-1 and MGD-2, Crassostrea gigas defensin (Cg-Def) is an original member of this family due to the presence of two extra cysteine residues
Summary
Animals and RT-PCR Amplification of Cg-def—Adult oysters, C. gigas, were purchased from a local oyster farm in Normandie (France) or Palavas (Gulf of Lion, France) and kept in seawater at 15 °C. Purification and Folding of Recombinant Cg-Def—His-tagged Cg-Def fusion protein was purified by affinity chromatography by incubating cell lysates with nickel-nitrilotriacetic acid resin (Novagen) at a ratio of 25:1 (v/v) in 6 M guanidine HCl, 20 mM Tris-HCl (pH 8.1) for 4 h at 4 °C. The Sep-Pak fraction was lyophilized, reconstituted in ultrapure water, and loaded onto a C8-reversed-phase high performance liquid chromatography (RP-HPLC) on an UP10C8 column (Interchrom modulocart uptisphere 10 C8, 250 ϫ 10 mm), and elution was performed using a 0 –55% acetonitrile gradient developed over 30 min at a flow rate of 5 ml/min. The relative expression ratio of Cg-def was calculated based on the crossing points deviation of each RT-PCR product of RNA extracted from stimulated oyster versus the appropriate control sample and expressed in comparison to the reference gene EF. The relative expression ratio of Cg-Def was calculated based on the delta-delta method for comparing relative expression results [28]
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