Identification, expression and activity analyses of five novel duck beta-defensins

Abstract

In the current study, five novel avian β-defensins (AvBDs) were identified and characterized in tissues from Peking ducks (Anas platyrhynchos). The nucleotide sequences of these cDNAs comprised 198 bp, 182 bp, 201 bp, 204 bp, and 168 bp, and encoded 65, 60, 66, 67, and 55 amino acids, respectively. Homology, characterization and comparison of these genes with AvBD from other avian species confirmed that they were Apl_AvBD1, 3, 5, 6, and 16. Recombinant AvBDs were produced and purified by expressing these genes in Escherichia coli. In addition, peptides were synthesized according to the respective AvBD sequences. Investigation of the antibacterial activity of the Apl_AvBDs showed that all of them exhibited antibacterial activity against all 12 bacteria investigated (P<0.05 or P<0.01). In addition, the antibacterial activity of all of the AvBDs against M. tetragenus and P. multocida decreased significantly in the presence of 150 mM NaCl (P<0.01). None of the AvBDs showed hemolytic activity. Consistent with their broad-spectrum antibacterial activity, the five novel Apl_AvBDs inhibited replication of duck hepatitis virus (DHV) in vitro significantly (P<0.05). The mRNA expression of all five Apl_AvBD in most tissues, including immune organs and the liver, was upregulated in response to DHV infection at different time points. These findings provide evidence that these defensins activate the immune response to combat microbial infection.

Figure 1. Deduced amino acid sequence alignment of the five novel avian β-defensins (AvBDs) from the duck.

Signal sequences of the Apl_AvBDs are italic. The conserved six cysteines (C) are framed. Dashes indicate no identical or conserved residues observed.

Figure 2. SDS–PAGE analysis of glutathione-S-transferase (GST)-tagged recombinant Apl_avian β-defensin (Apl_AvBD) proteins expressed in E. coli BL21 (DE3) cells.

(A) total protein from BL21 containing Apl_AvBD1, 3, 5, 6, 16, and GST with IPTG induction, respectively; (B) inclusion bodies with Apl_AvBD1, 3, 5, 6, 16, and GST respectively; (C) purified proteins of Apl_AvBD1, 3, 5, 6, 16, and GST with IPTG induction, respectively. IPTG, isopropyl-beta-D-thiogalactoside.

Figure 3. Antimicrobial activity of glutathione-S-transferase (GST), recombinant Apl_avian β-defensins (r Apl_AvBDs), and respective synthetic Apl_AvBDs (sApl_AvBDs) against bacteria.

(A) Gram-positive bacteria; (B) Gram-negative bacteria. Bactericidal activity was calculated as the percentage of colony counts of bacteria not exposed to antimicrobial peptides but subjected to the same experimental conditions. All studies were performed in three independent experiments with three replicates per experiment, and each bar represents the mean ± SD. The data were analyzed using SAS .

Figure 4. Effects of salinity on the antibacterial activity of Apl_avian β-defensins (Apl_AvBDs) against M. tetragenus or P. multocida.

(A) 25 µg/mL Apl_AvBDs; (B) 100 µg/mL Apl_AvBDs. Apl_AvBDs were incubated separately with either M. tetragenus or P. multocida for 3 h in the presence of 0, 50, 100, and 150 mM NaCl. All assays were performed in three independent experiments with three replicates per experiment, and each bar represents the mean ± SD. The data were analyzed using SAS .

Figure 5. Antiviral activities of glutathione-S-transferase (GST), and recombinant Apl_avian β-defensins (rApl_AvBDs) against duck hepatitis virus.

All assays were performed in three independent experiments, with five replicates per experiment, and each bar represents the mean ± SD. The data were analyzed using SAS , and P<0.05 is indicated by an asterisk “*”.

Figure 6. Hemolytic activities of duck avian β-defensins (AvBDs).

Freshly isolated duck red blood cells were incubated with different concentrations of AvBDs (0–500 µg/mL). Release of hemoglobin, as a measure of hemolysis, was measured at 405 nm. Release of hemoglobin upon addition of 1% Triton X-100 was set at 100%. The percentage of hemolysis was calculated as [(A _{405 nm, peptide} – A _{405 nm, PBS})/(A _{405 nm, 1% Triton X-100}– A _{405 nm, PBS})] × 100%. All assays were performed in three independent experiments, with three replicates per experiment, and each point is the mean ± SD. The data were analyzed using SAS .

Figure 7. Induction of mRNA of Apl_avian β-defensins (Apl_AvBDs) in tissues from ducks infected with duck hepatitis virus.

The cDNA copy number was measured by real time RT-PCR in tissues from five ducklings at 24, 32, 48, 72, and 96 h after infection, respectively. All assays were performed in three independent experiments, with three replicates per experiment, and each point is the mean ± SD. The data were analyzed using SAS .