RprDefB

Defensins represent an evolutionary ancient family of antimicrobial peptides, which played an undeniably important role in host defense. In the present study, a defensin isoform was identified and characterized from manila clam Ruditapes philippinarum (designed as Rpdef1α). Multiple alignments and phylogenetic analysis suggested that Rpdef1α belonged to the defensin family. Quantitative RT-PCR and immunohistochemical analysis revealed that Rpdef1α transcripts and the encoding peptide were dominantly expressed in the tissues of gills and mantle. After Vibrio anguillarum challenge, the Rpdef1α transcripts were significantly up-regulated in gills of clams. In addition, rRpdef1α not only showed broad-spectrum antimicrobial activities towards Vibrio species, but also inhibited the formation of bacterial biofilms. Knockdown of Rpdef1α transcripts caused significant increase in the cumulative mortality of manila clams post V. anguillarum challenge. Membrane integrity, scanning electron microscopy analysis and electrochemical assay indicated that rRpdef1α was capable of causing bacterial membrane permeabilization and then resulted in cell death. Moreover, phagocytosis and chemotactic ability of hemocytes could be significantly enhanced after incubation with rRpdef1α. Overall, these results suggested that Rpdef1α could act as both antibacterial agent and opsonin to defend against the invading microorganisms in manila clam R. philippinarum.

Bactericidal permeability-increasing protein (BPI) is an antimicrobial protein with potent endotoxin-neutralising activity and plays a crucial role in innate immunity against bacterial infection. In the present study, a bpi (designed as rpbpi) was identified and characterized from manila clam Ruditapes philippinarum. Multiple alignments and phylogenetic analysis suggested that rpbpi was a new member of the bpis family. In non-stimulated clams, rpbpi transcripts were ubiquitously expressed in all tested tissues with the highest expression level in hemocytes. After Vibrio anguillarum challenge, the expression levels of rpbpi mRNA in hemocytes were up-regulated significantly at 3 h and 48 h compared with that in the control, which were 4.01- and 19.10-fold (P < 0.05), respectively. The recombinant RpBPI (rRpBPI) showed high antibacterial activities against Gram-negative bacteria Escherichia coli and V. anguillarum, but not Staphylococcus aureus. Moreover, membrane integrity analysis revealed that rRpBPI increased the membrane permeability of Gram-negative bacteria, and then resulted in cell death. Overall, our results suggested that RpBPI played an important role in the elimination of invaded bacteria through membrane-disruptive activity.

Lysozyme is an antibacterial enzyme that is widely distributed in nature and plays an important role in the prevention of bacterial infections. In this study, a c-type lysozyme (designated as "RpCLYZ") was cloned and characterized from the manila clam, Ruditapes philippinarum. The full-length cDNA was 619 bp with an open reading frame (ORF) of 447 bp, and encoded a polypeptide of 148 amino acids with a calculated molecular mass of 17.0 kDa and an isoelectric point of 4.83. RpCLYZ was found to share high sequence similarity with c-type lysozymes from other invertebrates. The mRNA transcript of RpCLYZ was universally expressed in a wide range of tissues, especially in gills and mantle. Challenge with Vibrio anguillarum, significantly induced mRNA expression of RpCLYZ, which reached a maximum level 48 h after bacterial challenge. Recombinant RpCLYZ (rRpCLYZ) exhibited antibacterial activities against both Gram-positive and Gram-negative bacteria. Additionally, the optimal pH and temperature for rRpCLYZ activity were determined to be 4.5 and 20 °C, respectively. These results suggest that RpCLYZ participates in innate immune responses against bacterial invasion.