Investigation of avian defensins

Abstract

Banning the use of antibiotic growth promotors in animal feed has forced poultry breeding companies to select birds with improved disease resistance. One area of focus is the chicken innate immune system, which includes a family of avian β-defensins (AvBDs) that are synthesised by epithelia in response to microbial challenge. The aim of this thesis was to investigate the in vivo gene expression, antimicrobial activity (AMA) and mechanisms of action of AvBDs 6 and 9. Endpoint and qPCR were used to investigate AvBD6 and 9 gene expression in an array of epithelial tissues taken from day 7 and 35 broiler chickens reared in low and high hygiene conditions. The expression profiles of pro (interleukin-6: IL-6) and anti (Transforming Growth Factor β4:TGFβ4) inflammatory cytokine genes, and the chicken galectin-3 gene (CG3) were also examined. To explore AMA recombinant (r)AvBD peptides and their variants, including rAvBD9 3CA (lacking 3 of the 6 conserved cysteine (C) amino acids), rAvBD9 6CAG (lacking all the conserved C amino acids) and rAvBD9 W38G were synthesised, and their AMAs against E. coli and E. faecalis tested in vitro using time-kill assays. The structural properties and membrane interactions of the rAvBDs and custom synthesized linear (s) AvBD6 and 9 peptides were also investigated using circular dichroism (CD), liposome entrapped calcein leakage assays and peptide modelling. The in vivo gene expression analyses revealed that AvBD6 and 9, IL-6, TGFβ4 and CG3 were expressed in all the broiler chicken tissues examined. However, the data were compromised by the small bird numbers, and the variability in the tissue expression data between individual birds within a group resulted in no statistically significant trends associated with rearing environment being detected. The AMA data showed that the rAvBD6 and 9 peptides were antimicrobial against both Gram negative and positive microbes with rAvBD6 > rAvBD9 (using 100 μg/ml peptide, 76.4% (23.6±2.3 % survival) of E. coli were killed by rAvBD6 compared to 64.3% (35.7±7.2% survival) for rAvBD9). These data were potentially related to the physical properties of the peptide with AvBD6 being more cationic (+6.8) and hydrophobic than AvBD9 (+3.8), although modelling data also suggested that AvBD6 contained a hook-like foramen structure. The AMAs of the rAvBD9 and AvBD9 3CA peptides were not significantly different. Using 50 μg/ml peptide 49% (51±12% survival) and 60% (40±8% survival) of E. coli were killed compared to 53% (47±20% survival) and 69% (31±13% survival) killing for E. faecalis. Nu-PAGE data suggested the AMA potency of AvBD9 3CA was associated with dimer formation. At 50 μg/ml neither the rAvBD9 6CAG nor the synthetic linear AvBD9 peptides were active against the E.coli isolate. However, in the presence of proteinase inhibitor (Roche-1:1000 dilution), 45% E.coli BL21 killing (55±1% survival) was observed, which strongly supported the roles of the di-sulphide bonds in protecting the AvBD peptides against proteolysis. Substituting the C-terminal tryptophan (W) of rAvBD9 for a glycine (G) also resulted in a loss of AMA against bacteria (at 50 μg/ml 0% killing of E. coli and 15% (85.5 ±20.5% survival) killing of E. faecalis was detected). These data indicated that the C terminal W amino acid is also important for AvBD9 AMA. In the presence of SDS micelles mimicking the bacterial membrane synthetic AvBD1, 6 and 9 peptides showed increased α-helicity. Membrane leakage experiments using calcein-entrapped liposomes and synthetic peptides (1.5 μg/ml) showed sAvBD6 induced more leakage at 4 minutes than sAvBD9 (60.3±6.3% (n=4) versus 11.5% (n=1)). Although compromised by the lack of replicates these data suggested that AvBD9 may not function through membrane disruption suggesting other mechanisms including inhibition of nucleic acid synthesis and/or cell division. Membrane leakage experiments using sAvBD1 peptides modelling an AvBD1 SNP found in poultry showed NYH >SSY> NYY. Overall these data show AvBD6 and 9 are expressed in bird tissues and have AMA against gram negative and positive bacteria. The studies supported different mechanisms of action of the two defensins with AvBD6 causing membrane damage compared to AvBD9, which probably functions through disrupting intracellular systems. These data suggest that the AvBD peptides work in synergy in defending the epithelia and warn against poultry geneticists selecting individual AvBD genes for breeding purposes.