Defensin of the zebra mussel (Dreissena polymorpha): Molecular structure, in vitro expression, antimicrobial activity, and potential functions

A 409 bp full length defensin gene was cloned and sequenced based on an expressed sequence tag (EST) obtained from a normalized zebra mussel (Dreissena polymorpha) foot cDNA library developed in our laboratory. The D. polymorpha defensin (Dpd) gene encoded a peptide with 76 amino acid residues. The mature Dpd contains 54 amino acids with a fully functional insect defensin A domain. Homologue searching against GenBank database suggested that this Dpd was phylogenetically close to defensins from a group of insects with six conserved cysteine residues. Predicted with homology-modeling method, the three-dimensional structure of Dpd also demonstrated a significant similarity with insect defensin A. The recombinant Dpd was in vitro expressed through an Escherichia coli expression system. The antimicrobial activities of the refolded recombinant Dpd were found against the growth of Morganella sp., Plesiomonas shigelloides, Edwardsiella tarda, E. coli and Staphylococcus aureus aureus (ATCC12598) with the minimal inhibition concentration (MIC) 0.35, 0.43, 1.16, 6.46, and 30.39 μM, respectively. However, with less than 50 μM no detectable inhibition activities were observed against the other four Gram-negative bacteria (Aeromonas salmonicida salmonicida, Motile Aeromonas, Flavobacterium sp., Pseudomonas fluorescens, Shewanella putrifaciens), as well as the other Gram-positive bacteria (Bacillus megaterium ATCC14581, Carnobacterium maltaromaticum ATCC27865, Enterococcus faecalis ATCC19433, and Micrococcus leteus ATCC4698), and the yeast Saccharomyces cerevisiae. The RNA fluorescent in situ hybridization (FISH) of Dpd in the zebra mussel suggested that the Dpd was expressed in a variety of tissues, such as foot, retractor muscle, ctenidium, mantle, hemocytes, gonad, digestive gland, and intestine. By using the quantitative PCR, the expression level of Dpd in the zebra mussel foot was the highest, followed by the muscle. By comparing the amount of Dpd transcripts in the zebra mussel foot under two different byssogenesis conditions (byssogenesis and non-byssogenesis) with qPCR, we found that the higher expression levels of Dpd were always associated with the byssogenesis status from 2 to 4 days after the start of byssogenesis. Findings of this study suggest that the expression of the Dpd gene was upregulated in vivo by byssogeneis, and by hemocytes in vitro upon stimulation with microbial antigens.