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

Abstract

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.

A defensin from clam Venerupis philippinarum: Molecular characterization, localization, antibacterial activity, and mechanism of action

“…Allam et al (2006) reported a rapid increase (within 72 h) of circulating hemocytes in V. philippinarum after injection of Vibrio tapetis and V. anguillarum. In general, expression of marine mollusk defensin transcripts is known to be induced by bacterial challenges (Adhya et al, 2012;De Zoysa et al, 2010;Rosa et al, 2011;Zhao et al, 2007Zhao et al, , 2010. However, there are some reports of decreased transcriptional responses of mollusk defensins against bacterial infection.…”

The Effects of 4-Nonylphenol on the Immune Response of The Pacific Oyster, Crassostrea Gigas, Following Bacterial Infection (Vibrio Campbellii)

“…2A). This result was surprising given the strong Vibrio induced increase in Cg-bigdef1 reported by Rosa et al (2011) We also quantified Cg-bpi1, which encodes for the AMP bactericidalpermeability-increasing protein. BPI's activities are a result of its ability to permeabilize bacterial membranes (Gonzalez et al, 2007b) due to their affinity for binding to lipopolysaccharides found on the outer membrane of gram-negative bacteria (Elsbach & Weiss, 1998;Levy, 2000).…”

“…Defensins (Cg-defh1-2) are a prominent group of AMPs, which are also ubiquitously distributed across unicellular and multicellular organismal groups (Schmitt et al, 2010). Representatives of a separate family of 'big' defensins have also been characterized in C. gigas (Cg-bigdef1, Cgbigdef2 and Cg-bigdef3;Rosa et al, 2011). These 'big' defensins are a group of AMPs predominantly found in mollsucs, and are genetically and structurally more similar to vertebrate defensins than to the invertebrate defensins (Rosa at el., 2011).…”

“…Immediately following injection, oysters were placed back into their respective experimental tanks and held for 24 h (Macey et al 2008;Williams et al, 2009;Rosa et al, 2011) under their original 4-NP (or control) conditions. For the lysozyme activity measurements, we used sterile needles to collect roughly 200 μl of hemolymph directly from the adductor muscle at 3 h and 24 h post-injection (see 2.4 below).…”

“…Had we pooled our hemolymph samples, the hemocyte cDNA concentrations may have been sufficient to reliably quantify Cg-bigdef1 in our own hemocyte samples. That being said, Rosa et al (2011) also reported extreme variability in transcript abundances for Cg-bigdef1, Cg-bigdef2 and Cg-bigdef3 in hemocytes despite analyzing RNA from pooled (n=10 oysters) hemolymph samples. This variability has since been attributed to a high level of gene sequence polymorphisms in oysters (Hedgecock et al, 2004;Sauvage et al, 2007), which is particularly high for the Cg-defhs and Cg-bigdefs (Schmitt et al, 2013;Rosa et al, 2015).…”