Novel synthetic analogues of avian β-defensin-12: the role of charge, hydrophobicity, and disulfide bridges in biological functions

Abstract

Background: Avian 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.

Results: In 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.

Conclusions: While 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.

Keywords: Antimicrobial activity; Avian β-defensin analogues; Chemotactic activity; Disulfide bridges; Hydrophobicity; Net positive charge.

Fig. 1

The predicted three dimensional structures of AvBD-6 and AvBD-12. I-TASSER online service program was used to predict peptide structures. a Superimposition of the three dimensional structures of AvBD-12 and hBD-6. The CCR2 binding surface of hBD6 is highlighted in purple and the corresponding region in AvBD-12 is highlighted in yellow. b Superimposition of AvBD-12 and AvBD-6. c Distribution of positively and negatively charged amino acid residues in AvBD-12. d Distribution of positively charged amino acids in AvBD-6. Basic and acidic amino acids are highlighted in red and blue, respectively

Fig. 2

The predicted β2-β3 loop in AvBD-12A2 and AvBD-12A3. a Superimposition of AvBD-12A2 (green) and AvBD-12A3 (red) revealing the structural differences in the β2-β3 loop, a component of CCR2 binding domain. b Enlarged review of the β2-β3 loop in AvBD-12A2. The hydrogen bond between the -C = O group of F28 main chain and the -NH group of R29 side chain causes the arginine residue to fold back. c Enlarged review of the β2-β3 loop in AvBD-12A3. The CHO interactions between S27 -OH groups and the -CH groups on the aromatic ring of F28 result in an outward protrusion of R29 and a parallel twist of F28 aromatic ring. Distance: Å

Fig. 3

Antimicrobial activity of group 1 analogues. Bacteria (10⁵ CFU/ml) were incubated with peptides at various concentrations, ranging from 2 to 128 μg/ml at 37 °C for 2 h. Antimicrobial activity was presented as percent of killing compared to non-AvBD treated control. Antimicrobial activity of analogues against E. coli (a), P. aeruginosa (b), S. Typhimurium (c) and S. aureus (d). Data are means ± SD (n = 3). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Duncan’s test for multiple comparisons using software SPSS version 19.0 (IBM Corp., Armonk, NY). Asterisk indicates statistically significant difference between the analogues and AvBD-12 at the same concentrations (*p < 0.05). Solid line: average killing percent of 32 μg/ml of wild-type AvBD-12. Dash line: average killing percent of 4 μg/ml of wild-type AvBD-12

Fig. 4

Antimicrobial activity of group 2 analogues. Bacteria (10⁵ CFU/ml) were incubated with peptides at various concentrations, ranging from 2 to 128 μg/ml, at 37 °C for 2 h. Antimicrobial activity was presented as percent of killing compared to non-AvBD treated control. Antimicrobial activity of analogues AvBD-12A4 to A6 against E. coli (a), P. aeruginosa (b), S. Typhimurium (c) and S. aureus (d). Wild-type AvBD-6 and AvBD-12 were included as controls. Data are means ± SD (n = 3). Statistical analysis was performed using the one-way analysis of variance (ANOVA) followed by Duncan’s test for multiple comparisons using software SPSS version 19.0 (IBM Corp., Armonk, NY). Asterisks indicate statistically significant difference between analogues and AvBD-12 at the same concentrations (*p < 0.05). Solid line: average killing percent of 32 μg/ml of wild-type AvBD-12. Dash line: average killing percent of 4 μg/ml of wild-type AvBD-12

Fig. 5

Antimicrobial activity of hybrid peptide AvBD-12/6. Bacteria (10⁵ CFU/ml) were incubated with peptides at various concentrations, ranging from 2 to 128 μg/ml, at 37 °C for 2 h. Antimicrobial activity was presented as percent of killing compared to non-AvBD treated control. Antimicrobial activity of analogue AvBD-12/6 against E. coli (a), P. aeruginosa (b), S. Typhimurium (c) and S. aureus (d). Wild-type AvBD-6 and AvBD-12 were included as controls. Data are means ± SD (n = 3). Statistical analysis was performed using the one-way analysis of variance (ANOVA) followed by Duncan’s test for multiple comparisons using software SPSS version 19.0 (IBM Corp., Armonk, NY). Asterisks indicate statistically significant difference between the hybrid analogue and AvBD-12 at the same concentrations (*p < 0.05). Solid line: average killing percent of 32 μg/ml of wild-type AvBD-12. Dash line: average killing percent of 4 μg/ml of wild-type AvBD-12

Fig. 6

Effect of NaCl on the antimicrobial activity of group one AvBDs. Bacteria were treated with group one analogues in the presence of 5 mM, 50 mM, 100 mM or 150 mM NaCl. AvBDs were used at the following concentrations: 16 μg/ml against E. coli (a), 32 μg/ml against P. aeruginosa (b) and S. Typhimurium (c) and 64 μg/ml against S. aureus (d). These concentrations were chosen to ensure more than 50% of killing of inoculum by majority of analogues. Results are expressed as percent of killing compared to the no-salt control. Data shown are means ± SD (n = 3). Statistical analysis was performed using the one-way analysis of variance (ANOVA) followed by Duncan’s test for multiple comparisons using software SPSS version 19.0 (IBM Corp., Armonk, NY). Asterisks indicate statistically significant difference among different treatment groups (*p < 0.05, **p < 0.01)

Fig. 7

Effect of NaCl on the antimicrobial activity of group two analogues. Bacteria were treated with group two analogues in the presence of 5 mM, 50 mM, 100 mM or 150 mM NaCl. AvBDs were used at the following concentrations: 16 μg/ml against E. coli (a), 32 μg/ml against P. aeruginosa (b) and S. Typhimurium (c) and 64 μg/ml against S. aureus (d). These concentrations were chosen to ensure more than 50% of killing of inoculum by majority of analogues. Results are expressed as percent of killing compared to the no-salt control. Data shown are means ± SD (n = 3). Statistical analysis was performed using the one-way analysis of variance (ANOVA) followed by Duncan’s test for multiple comparisons using software SPSS version 19.0 (IBM Corp., Armonk, NY). Asterisks indicate statistically significant difference among different treatment groups (*p < 0.05, **p < 0.01)

Fig. 8

Effect of NaCl on the antimicrobial activity activity of group three analogue AvBD-12/6. Bacteria were treated with AvBDs in the presence of 5 mM, 50 mM, 100 mM or 150 mM NaCl. AvBDs were used at the following concentrations: 16 μg/ml against E. coli (a), 32 μg/ml against P. aeruginosa (b) and S. Typhimurium (c) and 64 μg/ml against S. aureus (d). These concentrations were chosen to ensure more than 50% of killing of inoculum by majority of analogues. Results are expressed as percent of killing compared to the no-salt control. Data shown are means ± SD (n = 3). Statistical analysis was performed using the one-way analysis of variance (ANOVA) followed by Duncan’s test for multiple comparisons using software SPSS version 19.0 (IBM Corp., Armonk, NY). Asterisks indicate statistically significant difference among different treatment groups (*p < 0.05, **p < 0.01)

Fig. 9

Chemotactic activity of AvBD-12 analogues for CCR2 transfected CHO-K1 cells and mouse immature dendritic JAWSII cells. Migration of CCR2 transfected CHO-K1 cells (a-c) and mouse immature dendritic JAWSII cells (d-f). The results are expressed as chemotactic index (C.I.): the number of migrated cells induced by AvBD analogues / the number of migrated cells in response to chemotactic buffer. Data are means ± SD (n = 5). Statistical analysis was performed using the one-way analysis of variance (ANOVA) followed by Duncan’s test for multiple comparisons using software SPSS version 19.0 (IBM Corp., Armonk, NY). Asterisks indicate statistically significant difference between analogues and wild-type AvBD-12 (*p < 0.05, **p < 0.01)

Fig. 10

Scanning electron microscopy (SEM) of S. Typhimurium treated with AvBDs. Mid-logarithmic-phase S. Typhimurium cells (10⁸ CFU) were incubated with AvBDs at a final concentration of 1 × MIC-ls for 30 min. a AvBD-12A3, b AvBD-12/6, c AvBD-12, d AvBD-6, e Stationary phase bacteria, f Mid-logarithmic-phase S. Typhimurium treated with PBS. Arrow 1, membrane damage. Arrow 2, cell deformation. Arrow 3, cell death in stationary-phase culture. Arrow 4, a normal cell in mid-logarithmic-phase culture. Scale bar: 5 μm