Abstract
BackgroundAvian β-defensins (AvBD) possess broad-spectrum antimicrobial, LPS neutralizing and chemotactic properties. AvBD-12 is a chemoattractant for avian immune cells and mammalian dendritic cells (JAWSII) — a unique feature that is relevant to the applications of AvBDs as chemotherapeutic agents in mammalian hosts. To identify the structural components essential to various biological functions, we have designed and evaluated seven AvBD analogues.ResultsIn the first group of analogues, the three conserved disulfide bridges were eliminated by replacing cysteines with alanine and serine residues, peptide hydrophobicity and charge were increased by changing negatively charged amino acid residues to hydrophobic (AvBD-12A1) or positively charged residues (AvBD-12A2 and AvBD-12A3). All three analogues in this group showed improved antimicrobial activity, though AvBD-12A3, with a net positive charge of +9, hydrophobicity of 40% and a predicted CCR2 binding domain, was the most potent antimicrobial peptide. AvBD-12A3 also retained more than 50% of wild type chemotactic activity. In the second group of analogues (AvBD-12A4 to AvBD-12A6), one to three disulfide bridges were removed via substitution of cysteines with isosteric amino acids. Their antimicrobial activity was compromised and chemotactic activity abolished. The third type of analogue was a hybrid that had the backbone of AvBD-12 and positively charged amino acid residues AvBD-6. The antimicrobial and chemotactic activities of the hybrid resembled that of AvBD-6 and AvBD-12, respectively.ConclusionsWhile the net positive charge and charge distribution have a dominating effect on the antimicrobial potency of AvBDs, the three conserved disulfide bridges are essential to the chemotactic property and the maximum antimicrobial activity. Analogue AvBD-12A3 with a high net positive charge, a moderate degree of hydrophobicity and a CCR2-binding domain can serve as a template for the design of novel antimicrobial peptides with chemotactic property and salt resistance.
Highlights
Avian β-defensins (AvBD) possess broad-spectrum antimicrobial, LPS neutralizing and chemotactic properties
Additional H3V8L21I29 for D3D8E21E29 substitutions further increased the hydrophobicity to 53% and the net positive charge from +1 (AvBD-12) to +5 (AvBD-12A1)
These results collectively suggest that all three disulfide bridges are needed in the natural form of Avian beta-defensin (AvBD) to maintain tertiary structural features critical to interacting with microbial surface components and CCRs on immune cells
Summary
Avian β-defensins (AvBD) possess broad-spectrum antimicrobial, LPS neutralizing and chemotactic properties. Beta-defensins are small, cationic, antimicrobial peptides found in different living organisms [6,7,8,9]. Beta-defensins possess various biological properties, including broad-spectrum microbicidal activity, neutralization of LPS, activation of macrophages and dendritic cells, chemoattraction of dendritic cells, monocytes, and T-lymphocytes to the site of infection [7, 10, 12]. The chemotactic function of βdefensins is mediated by CC-chemokine receptors, such as CCR2 and CCR6 [16,17,18] These natural antimicrobial peptides with complex mechanisms of action represent potentially a novel class of antimicrobials [13, 19]. Several challenges must be addressed in order to develop β-defensins for therapeutic use which include retaining the biological activities under physiological conditions and the ease of production and purification of recombinant or synthetic peptides [20, 21]
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