Chicken AvBD2

Numerous β-defensins have been identified in birds, and the potential use of these peptides as alternatives to antibiotics has been proposed, in particular to fight antibiotic-resistant and zoonotic bacterial species. Little is known about the mechanism of antibacterial activity of avian β-defensins, and this study was carried out to obtain initial insights into the involvement of structural features or specific residues in the antimicrobial activity of chicken AvBD2. Chicken AvBD2 and its enantiomeric counterpart were chemically synthesized. Peptide elongation and oxidative folding were both optimized. The similar antimicrobial activity measured for both L- and D-proteins clearly indicates that there is no chiral partner. Therefore, the bacterial membrane is in all likelihood the primary target. Moreover, this work indicates that the three-dimensional fold is required for an optimal antimicrobial activity, in particular for gram-positive bacterial strains. The three-dimensional NMR structure of chicken AvBD2 defensin displays the structural three-stranded antiparallel β-sheet characteristic of β-defensins. The surface of the molecule does not display any amphipathic character. In light of this new structure and of the king penguin AvBD103b defensin structure, the consensus sequence of the avian β-defensin family was analyzed. Well conserved residues were highlighted, and the potential strategic role of the lysine 31 residue of AvBD2 was emphasized. The synthetic AvBD2-K31A variant displayed substantial N-terminal structural modifications and a dramatic decrease in activity. Taken together, these results demonstrate the structural as well as the functional role of the critical lysine 31 residue in antimicrobial activity.

Beta defensins (BD) are cysteine rich, cationic antimicrobial peptides (AMP) produced mainly by epithelial and myeloid cells such as neutrophils. In birds, the neutrophil equivalent heterophils produce avian beta defensins (AvBD) of which AvBD2 is the major isoform. Heterophils recognize pathogens or their derived products through a series of pattern recognition receptors called toll-like receptors (TLR) leading to their antimicrobial activities. This work is the first report of TLR modulation of AvBD2 expression in chickens. To measure the effect of TLR activation on AvBD2 production, the heterophils were cultured with different TLR agonists for 6h. Modulation of AvBD2 levels by TLR activation was measured using direct MALDI mass spectrometry without stable isotopic labeling or chromatographic separation. Chemical modification of the conditioned media was performed using reduction/alkylation with dithiothreitol/iodoacetamide to distinguish TLR treated AvBD2 (reduced/alkylated) from controls (non-reduced). Changes in corrected ion intensity ratios were assumed to reflect AvBD2 modulation in heterophils upon activation with different TLR agonists. In general, TLR agonists increased AvBD2 production with LPS showing the greatest induction and CpG-ODN showing little or no effect. These data show that the direct MALDI-MS coupled with reduction/alkylation may provide a rapid relative quantitative approach to the measurement of agonist-induced differential expression of AvBD2.

The cationic, cysteine-rich peptides called beta-defensins play a major role in the innate immune response. Here, we describe the identification and characterization of the duck beta-defensin-2 homologue, Anas platyrhynchos avian beta-defensin 2 (Apl_AvBD2). The 195 base pair open reading frame (ORF) of Apl_AvBD2 has 83% identity with Gga_AvBD2 (chicken) and 85% identity with Mga_AvBD2 (turkey) at nucleotide level. The gene corresponding to the coding region is comprised of three exons and two introns in both Apl_AvBD2 and Gga_AvBD2. The predicted secondary structure of Apl_AvBD2 has the classical "beta-defensin core motif" formed by the beta-sheet rich structure. Apart from mild expression in tissues like kidney, lung, brain, bursa of Fabricious and ovary, Apl_AvBD2 mRNA show a very high level constitutive expression in bone marrow and spleen, indicating that it is a myeloid defensin. Purified recombinant Apl_AvBD2 demonstrated in vitro antibacterial activity against both Gram-positive and Gram-negative bacteria, with a minimum bactericidal concentration (MBC) of 3.7 microM against Micrococcus luteus NCIM 2871 and Escherichia coli NCIM 2685, and of 2.2 microM against Reimerella anatipestifer. The immunomodulatory potential of Apl_AvBD2 was shown by chemotaxis of DT-40 chicken B-lymphocytes. The widespread tissue distribution and the potent bactericidal and chemotactic activity make Apl_AvBD2 an important molecule in the innate immune response in ducks. It may play a vital role in the immune response of these birds against bacterial and viral pathogens.