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

Design and synthesis of peptides

The three dimensional structures of AvBDs were predicted by using the I-TASSER program (http://zhanglab.ccmb.med.umich.edu/I-TASSER). The distribution of positively charged amino acids were evaluated using PyMOL (https://www.pymol.org/). Group 1 analogues, including AvBD-12A1, A2 and A3 were linear peptides, in which the six cysteines (C¹C²C³C⁴C⁵C⁶ or C5C12C17C27C34C35) were replaced with structurally similar amino acid residues (alanine and serine) as follows: AvBD-12A1: A5A12A17A27A34A35, AvBD-12A2: S5S12S17A27A34A35, and AvBD-12A3: A5A12A17S27S34S35. Additional modifications were introduced to these analogues to alter peptide hydrophobicity and charge. In AvBD-12A1, the four negatively charged amino acid residues (D3D8E21E29) were replaced with one polar and three hydrophobic amino acid residues (H3V8L21I29). In AvBD-12A2 and AvBD-12A3, D3D8E21E29 were substituted with positively charged residues R3K8K21R29. Group 2 analogues, including AvBD-12A4, AvBD-12A5 and AvBD-12A6 had reduced number of disulfide bridges without any additional modifications. To remove disulfide bridges, the cysteine residues (C¹C²C³C⁴C⁵C⁶ or C5C12C17C27C34C35) of AvBD-12 were replaced with isosteric α-aminobutyric acids (Abu, U) to create AvBD-12A4 (U5C12C17C27U34C35), A5 (U5U12C17U27U34C35), and A6 (U5U12U17U27U34U35). Group 3 included a single hybrid peptide, namely AvBD-12/6. This analogue was designed using the backbone of AvBD-12, in which the negatively charged amino acid residues (D3D8E21E29) of AvBD-12 were replaced with amino acids (H3Q8Y21S29) of AvBD-6 at the corresponding positions. The hydrophobicity and charge of AvBD analogues at neutral pH were calculated using Peptide 2.0 (http://peptide2.com) and Peptide property calculator (PepCalc.com), respectively.

All peptides were custom synthesized using the standard solid phase 9-fluorenylmethoxycarbonyl (Fmoc) method and purified by reverse phase high performance liquid chromatography (RP-HPLC) (Lifetein, Hillsborough, NJ). The analogues AvBD-12A4, −A5, AvBD-12/6, AvBD-6 and AvBD-12 with varying numbers of cysteine residues were subjected to oxidative folding as described previously [17]. Electrospray ionization mass spectrometry (ESI-MS) was performed to confirm the correct formation of disulfide bridges between Cys¹-Cys⁵, Cys²-Cys⁴ and Cys³-Cys⁶. The purity of the synthetic AvBD analogues was over 98.5% as verified by liquid chromatography-mass spectrometry (LC-MS) (Lifetein, Hillsborough, NJ).

Antimicrobial activity of AvBD analogues

Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Salmonella enteric serovar Typhimurium (ATCC 14028) and Staphylococcus aureus (ATCC 29213) were used to assess the antimicrobial activity of AvBD analogues. All bacterial strains were grown on Luria-Bertani (LB) agar plates or Trypticase Soy Agar with 5% Sheep Blood (TSA, Thermo Fisher Scientific) plates at 37 °C. Antimicrobial activity was determined by a colony counting assay [21, 26]. In brief, bacteria were resuspended in 100-fold diluted Mueller Hinton II broth with 5 mM NaCl (minimal growth medium) to obtain a final bacterial concentration of 2 × 10⁵ CFU/ml. Twenty-five microliters of bacterial suspension and 25 μl of AvBD analogue solution were mixed in the wells of a 96-well polypropylene microtiter plate (Nunc™, Thermo Fisher Scientific). The final peptide concentrations were 2, 4, 8, 16, 32, 64 and 128 μg/ml. The minimal growth medium without AvBD peptide was included as a negative control. The bacterial-peptide mixtures were incubated at 37 °C for 2 h, ten-fold serially diluted and plated on LB agar plates. The numbers of bacteria colonies were enumerated after 16 h of incubation at 37 °C. Antimicrobial activity was expressed as percent of killing using the following formula: (CFU_control - CFU_treated) / CFU_control × 100%. To assess the resistance of AvBD analogues to sodium chloride (NaCl), antimicrobial assays were carried out in the presence of 5 mM, 50 mM, 100 mM or 150 mM NaCl. All assays were performed in triplicate.

Minimum inhibitory concentrations of AvBD analogues

The minimum inhibitory concentrations (MIC) of AvBDs were determined according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [27, 28]. The Muller Hinton II broth used in standard MIC assays contained 20–25 mg/L of calcium and 10–12.5 mg/L of magnesium. Modified MICs of AvBDs were also determined under a low-salt and low-nutrient condition by using 100× diluted Muller Hinton II broth containing 0.2 mg/L of calcium, 0.1 mg/L magnesium and 5 mM (292 mg/L) NaCl. MIC obtained under the low-salt and low-nutrient condition was referred to as MIC-ls. AvBD peptides were two-fold serially diluted (2 to 256 μg/ml) in a 96-well microtiter plate. An equal volume (μl) of bacterial suspension was added to each well of the plate. The final bacterial concentration in the wells was 5 × 10⁵ CFU/ml. The plate was incubated at 37 °C for 24 h and the lowest concentration that completely prevented visible bacteria growth was recorded. To complement MIC-ls assays, the minimum bactericidal concentrations (MBC) were evaluated by sub-culturing the contents of the first two clear wells obtained in the MIC-ls assay onto LB agar plates. All assays were conducted in triplicate. The lowest peptide concentration inhibiting more than 99% of bacterial growth was defined as MBC-ls. AvBD analogues were regarded as bactericidal if the MBC was no more than four times the MIC [29].

Cell cytotoxicity assay

Chicken macrophage cell lines MQ-NCSU and HD11 were maintained in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS), 2% chicken serum, 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich) at 37 °C in humidified air with 5% CO₂. CHO-K1 cells were cultured in the same media without 2% chicken serum. For CCR2-transfected CHO-K1 cells, the medium was supplemented with 500 μg/ml G418 was added (Sigma-Aldrich) [26]. Murine immature dendritic cell line JAWSII (ATCC CRL-11904™) was cultured in Alpha minimum essential medium containing 4 mM L-glutamine, 1 mM sodium pyruvate, 5 ng/ml murine Granulocyte macrophage colony-stimulating factor (GM-CSF), 20% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in humidified air with 5% CO₂. The cell cytotoxicity was determined using the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide metabolic activity assay according to the manufacturer’s instruction (Thermo Fisher Scientific). Briefly, cells (5 × 10³ cells/well) were seeded in 96-well tissue culture plates, incubated overnight and treated with AvBDs at concentrations of 4, 16, 64 and 256 μg/ml for 4, 12, 24 and 48 h at 37 °C. After treatment, 20 μl of 12 mM MTT solution was added to each well. The plate was incubated for 4 h and read at 540 nm. Viability was expressed as percentage of viable cells relative to the untreated control. The experiments were performed in triplicate.

Chemotaxis assay

Migration of JAWSII and CCR2-CHO-K1 cells in response to AvBD-12 analogues was determined using a 48-well microchemotaxis chamber technique as previously described [30]. Chemotaxis buffer (Minimum Essential Medium containing 0.1% BSA, 100U/ml penicillin, and 100 μg/ml streptomycin) and bacterial peptide N-Formyl-methionyl-leucyl-phenylalanine (fMLF, Sigma-Aldrich) were included as negative and positive controls, respectively. The results were presented as chemotactic index (C.I.). C.I. = number of migrated cells induced by AvBDs / number of migrated cells induced by chemotactic buffer. The assay was repeated five times.

Scanning electron microscopy (SEM)

SEM was performed according to the procedure described by Cobo et al. [31]. S. Typhimurium was cultured in Mueller-Hinton (MH) broth to mid-log phase and harvested by centrifugation at 5000 g for 10 min. Cell pellets were washed twice with 10 mM PBS and resuspended at a final number of 10⁸ CFU. The cell suspension was incubated with 1 × MIC-ls of AvBD-12A3, AvBD-12/6, wild-type AvBD-6 and AvBD-12 at 37 °C for 30 min. Bacterial pellets were fixed in 500 ml of 2.5% (v/v) glutaraldehyde in 0.2 M cacodylate buffer at 4 °C overnight, washed twice with 0.2 M cacodylate buffer and dehydrated through ethanol gradient (30%, 50%, 70%, 90%, 100% and again 100%) for 15 min in each gradient. The samples were transferred into a mixture (1:1, v/v) of ethanol and tertiary butanol and then pure tertiary butanol for 20 min each. After gold coating, the specimens were observed using a scanning electron microscope (Hitachi S-4700, Japan).

Statistical analysis

Data were presented as the means ± standard deviation (SD). Differences between groups were analyzed 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). Differences at p < 0.05 level were considered statistically significant, and at p < 0.01 level were considered extremely significant.

Structural characteristics of AvBD-6 and AvBD-12 and their analogues

Substitutions of C¹C²C³C⁴C⁵C⁶ (or C5C12C17C27C34C35) by A5A12A17A27A34A35 (AvBD-12A1) not only eliminated the three disulfide bridges, but also elevated the hydrophobicity from 33% (AvBD-12) to 47%. Additional H3V8L21I29 for D3D8E21E29 substitutions further increased the hydrophobicity to 53% and the net positive charge from +1 (AvBD-12) to +5 (AvBD-12A1). Changes from C¹C²C³C⁴C⁵C⁶ to S5S12S17A27A34A35 (AvBD-12A2) or to A5A12A17S27S34S35 (AvBD-12A3) and changes from D3D8E21E29 to R3K8K21R29 (AvBD-12A2 and A3) eliminated all three disulfide bridges and increased the peptide hydrophobicity from 33% to 40% and the net positive charge from +1 to +9. AvBD-12A4, A5, and A6 respectively lost disulfide bridge(s) C^1–5; C^1–5 and C^2–4; C^1–5, C^2–4, and C^3–6. C to U changes did not affect the peptide hydrophobicity and charge. The hybrid AvBD-12/6 with the backbone of AvBD-12 and the positively charged amino acids (H3Q8Y21S29) of AvBD-6 retained parent peptides’ hydrophobicity (33%) and an intermediate charge (+5), compared to AvBD-6 (+7) and AvBD-12 (+1). The sequence, charge, hydrophobicity and number of disulfide bridges of analogues and wild-type AvBD-12 and AvBD-6 were listed in Table 1.

^aDisulfide bridges (S-S) between C^1–5, C^2–4, C^3–6

^bU: α-aminobutyric acid

^cAcidic amino acids, basic amino acids, and cysteines are highlighted in blue, red, and green color, respectively. C¹C²C³C⁴C⁵C⁶ or C5 C12 C17 C27 C34 C35

Superimposition of the predicted three dimensional structures of AvBD-12 and the structure of human β-defensin 6 (hBD6) revealed a similar N-terminal α-helix and an adjacent β2-β3 loop, in addition to the conserved internal β-sheet domains (Fig. 1a). The α-helix and β2-β3 loop have been identified by NMR spectroscopy as a contiguous binding surface for human CCR2 [32]. A comparison of the predicted three dimensional structures of AvBD-6 and AvBD-12 showed only the β2-β3 loop in AvBD-6. AvBD-6 had an N-terminal coil turn instead of an α-helix. Peptide charge distribution analysis indicated that positively charged amino acid residues (H4R7R10R38R40) in the N- and C-termini of AvBD-6 formed a cluster whereas positively charged residues (H7R9K36) of AvBD-12 were separated by negatively charged residues (Fig. 1d). The N-terminal α-helix and the β2-β3 loop were also seen in the predicted structures of AvBD-12A2 and AvBD-12A3. However, differences in the β2-β3 loop between AvBD-12A2 and A3 were identified (Fig. 2a). In AvBD-12A2, the -C = O group in the main chain of F28 formed a hydrogen bond with the -NH group in the side chain of R29, resulting in the fold-back of R29 sidechain (Fig. 2b). In AvBD-12A3, there was no hydrogen bond formation between R29 and F28. Instead, there are C-H/O interactions between the -CH groups in the aromatic π ring of F28 and the -C = O group of F28 as well as the -CH group in the side chain of S27, similar to what was reported previously [33]. Consequently, the side chain of R29 protruded towards the surface of the peptide and the aromatic ring of F28 in AvBD-12A3 turned in parallel with R29 side chain (Fig. 2c).

Antimicrobial activity

Group 1 analogues with increased net positive charge and hydrophobicity were significantly more effective (p < 0.05) than the parent peptide AvBD-12 in killing E. coli, S. Typhimurium, P. aeruginosa, and S. aureus (Fig. 3). At 16 μg/ml, AvBD-12, AvBD-12A1, AvBD-12A2 and AvBD-12A3 killed 74.4%, 88.6%, 100%, 100% of E. coli; 47.3%, 95.6%, 93.9%, and 93.9% S. Typhimurium; 35.5%, 72.3%, 98%, and 100% of P. aeruginosa; and 52.9%, 77.2%, 83.1%, and 91.7% of S. aureus, respectively. AvBD-12A2 and A3 with a net positive charge of +9 and hydrophobicity of 40% were more effective than AvBD-12A1 (charge = +5, hydrophobicity = 53%). AvBD-12A2 and AvBD-12A3 which had identical charge and hydrophobicity but altered locations of alanine/serine residues exhibited different killing activities against E. coli or P. aeruginosa (Fig. 3). The bactericidal potency of group-1 analogues can be ranked as AvBD-12A3 > AvBD-12A2 > AvBD-12A1 > AvBD-12. Of the bacterial species tested, E. coli and P. aeruginosa were more susceptible than S. Typhimurium and S. aureus to AvBD-12A3 at medium concentrations, ranging from 8 μg/ml to 32 μg/ml (Fig. 3).

Group 2 analogues, including AvBD12A4, AvBD-12A5, and AvBD-12A6 showed similar killing activity to that of AvBD-12 (Fig. 4). One exception was AvBD-12A4 was an exception that lost one disulfide bridge (C¹-C⁵ or C5-C34) was shown to have weaker action than AvBD-12 against S. Typhimurium (p < 0.05). Group 3 or the hybrid analogue, AvBD-12/6 with the backbone of AvBD-12 and positively charged amino acid residues of AvBD-6 exhibited similar killing activities to that of AvBD-6 (Fig. 5).

Minimum inhibitory concentration

The MIC-ls of AvBD-12A1, AvBD-12A2 and AvBD-12A3 against E. coli, S. Typhimurium, and P. aeruginosa were 2 to 16-fold below that of AvBD-12, confirming the improved antimicrobial property of these analogues. The MIC-ls of these analogues against P. aeruginosa were 2 to 8-fold below that of AvBD-6. The ratio of MBC-ls/MIC-ls was equal to or below 4:1 for AvBD-12A2 and AvBD-12A3 against the three Gram negative bacterial species tested, suggesting a bactericidal action [29]. The MIC-ls of AvBD-12A4, AvBD-12A5, and AvBD-12A6 with 2, 1 and 0 disulfide bridges, respectively, were higher than that of AvBD-12 and AvBD-6, indicating that removal of disulfide bridges compromised AvBD’s antimicrobial function.

The MIC-ls of AvBD analogues was negatively correlated with the net positive charge. The correlation co-efficiencies (r) for E. coli, S. Typhimurium, P. aeruginosa, and S. aureus were −0.7388, p < 0.05; −0.8545, p < 0.01; −0.8545, p < 0.01; and −0.8727, p < 0.01, respectively. Although increasing hydrophobicity also resulted in improved antimicrobial potency, a positive correlation between hydrophobicity and antimicrobial activity was not found.

Susceptibility to NaCl

The impact of NaCl on the killing activity of AvBDs was assessed at NaCl concentrations ranging from 5 to 150 mM. Increasing NaCl concentration had less adverse impact on the killing activity of AvBD-12A2 and AvBD-12A3 which had a higher net positive charge (+9) and hydrophobicity (40%) than AvBD-12 (Fig. 6). AvBD-12A1, the most hydrophobic (53%) analogue, showed similar or increased susceptibility to NaCl, compared to AvBD-12 (Fig. 6). Susceptibility to NaCl was also influenced by the bacterial species under investigation. At 150 mM NaCl, AvBD-12A2 and AvBD-12A3 retained approximately 80% of killing potency against E. coli and P. aeruginosa, but only 30% to 60% of killing activity against S. Typhimurium and S. aureus (Fig. 6). AvBD-12A4, AvBD-12A5, and AvBD-12A6 with fewer disulfide bridges were equally or more susceptible to NaCl than AvBD-12 (Fig. 7). AvBD-12/6 with the backbone of AvBD-12 and increased net positive charge (+5) resembled AvBD-6 instead of AvBD-12 (Fig. 8).

Cytotoxicity to host cells

Cell cytotoxicity of most potent analogue AvBD-12A3 (+9) and the hybrid analogue AvBD-12/6 (+5) was determined. Exposure of chicken macrophage cell lines HD11 and MQ-NCSU, murine dendritic cell line JAWSII and CHO-K1 cells to AvBDs at concentrations of 4, 16, 64, 256 μg/ml for 4, 12, 24, and 48 h did not affect cell variability. Data on the highest peptide concentration (256 μg/ml) at various exposure times were presented in Additional file 1: Figure S1. The results were consistent with our previous findings that AvBD-6 and AvBD-12 were non-cytotoxic to avian and mammalian cell lines [26].

Chemotactic activity

Group 1 analogues AvBD-12A1, AvBD-12A2 and AvBS-12A3 showed minimal chemotactic activity for CCR2-CHO cells (Fig. 9a). However, AvBD-12A2 and AvBD-12A3 at 64 μg/ml demonstrated mild (C.I. = 2.37; 35.9% of wild type) and modest (C.I. = 3.74; 56.6% of wild type) chemotactic activity, respectively, for JAWSII cells (Fig. 9b). Analogues AvBD-12A4 with two disulfide bridges had mild chemotactic activity for CCR2-CHO (C.I. = 1.48 to 2.18) and JAWSII cells (C.I. = 1.05 to 1.86) which were significantly below the chemotactic index of parent peptide AvBD-12 (p < 0.01, Fig. 9c and d). AvBD-12A5 with one disulfide bridge and AvBD-12A6 with zero disulfide bridges lost their chemotactic activity for both CCR2-CHO-K1 and JAWSII cells (Fig. 9c and d). The hybrid AvBD-12/6 retained the chemotactic function of the backbone peptide AvBD-12 (Fig. 9e and f).

SEM observation

Mid-logarithmic-phase S. Typhimurium bacteria treated with AvBD-12A3 (Fig. 10a), AvBD-12/6 (Fig. 10b), AvBD-12 (Fig. 10c) and AvBD-6 (Fig. 10d) displayed cell membrane damage (arrow 1) and cell deformation (arrow 2). Mid-logarithmic-phase S. Typhimurium cells treated with PBS showed normal size and intact structure (Fig. 10f, arrow 4). Cell death in stationary-phase culture (Fig. 10e, arrow 3) showed loss of intracellular content and uniform membrane structure.