Antimicrobial activity of DRGN-1

To investigate whether DRGN-1 could exert antimicrobial effects, we tested the activity of CAMPs against the gram-negative Pseudomonas aeruginosa, Francisella novicida and Burkholderia thailandensis and the gram-positive Staphylococcus aureus. It should be noted that VK25 is a histone H1-derived peptide from Komodo, and histone peptides are generally weakly active or inactive under minimal inhibitory concentration (MIC) conditions, but may still have activity under low-salt, phosphate buffer conditions (EC 50 ).16, 17 The results showed that DRGN-1 demonstrated the antimicrobial activity in an EC 50 assay against the gram-negative bacterium P. aeruginosa (but not F. novicida or B. thailandensis) as well as the gram-positive bacterium S. aureus within the range of 0.77–7.1 µg/ml (0.50–4.62 µM). This was compared to VK25, which was significantly less active with anti-Pseudomonas activity in the range of 25–30 µg/ml and anti-Staphylococcus activity of more than 100 µg/ml (Table 1). These peptides do not show antimicrobial activity in full broth under MIC conditions, as is known for many other peptides, including the human cathelicidin LL-37.17, 18

Table 1 Antimicrobial (EC 50 ) activity of DRGN-1, VK25 and LL-37 against a panel of gram-positive and gram-negative bacteria Full size table

Anti-biofilm activity of DRGN-1

Next, the ability of DRGN-1 to prevent the formation of biofilm after 18 h was examined by crystal violet staining and confocal microscopy (Fig. 1). When cultured in a polystyrene tube under shaking conditions, P. aeruginosa and S. aureus form biofilms along the sidewall of the culture tube, which was strongly inhibited by DRGN-1 in a concentration-dependent manner (Fig. 1a, b). For confocal imaging, DRGN-1 (final concentration, 25 μg/ml) was incubated with bacteria overnight in the wells of a chamber slide. P. aeruginosa Green-fluorescent protein (GFP)+ and S. aureus Red-fluorescent protein (RFP)+ were used for these studies.19 The cells were incubated for 24 h in tryptic soy broth (TSB) medium in the presence of DRGN-1. The next day, the wells were rinsed and the development of a biofilm was compared with a control well (without DRGN-1). Biofilms of both P. aeruginosa and S. aureus were significantly reduced by DRGN-1. In mixed-culture biofilms grown for 24 h without treatment, P. aeruginosa significantly outgrows S. aureus,19 thus the appropriate starting ratio was established as a 99:1 ratio S. aureus to P. aeruginosa. Treatment with DRGN-1 had a significant inhibitory effect on the mixed bacterial cultures in contrast with untreated control.

Fig. 1 Anti-biofilm activity of DRGN-1. a DRGN-1 anti-biofilm activity against S. aureus using three interdependent methods. The activity was analyzed by crystal violet staining of biofilm in culture tube at 24 h. b DRGN-1 anti-biofilm activity against P. aeruginosa. Pictures (right inlet in a and b) are a representative biofilm stained with crystal violet. P values were calculated using a two-tailed t test (assuming unequal variances) comparing test strains to untreated control (*P < 0.05; **P < 0.01; ***P < 0.001). c Biofilm evaluation by confocal microscopy. Representative images of biofilms of P. aeruginosa PAO1-pTDK-GFP expressing GFP, S. aureus SH1000 pAH9-RFP expressing RFP or mixed cultures exposed to PBS (control) or DRGN-1 (25 µg/ml) for 24 h. DRGN-1-exposed biofilms are thinner and more sparse than untreated controls. Scale bar: 20 µm Full size image

Ability of DRGN-1 to affect P. aeruginosa-infected HEKa keratinocytes

To gain insight into the ability of DRGN-1 to exert activity against internalized P. aeruginosa, HEKa keratinocytes were infected by P. aeruginosa ATCC 9027, and subsequently treated with DRGN-1 or VK25. As highlighted in Fig. 2, the killing of infected keratinocytes was pronounced 2 h after their exposure to the bacteria, presumably due to P. aeruginosa-induced apoptotic cell death, which was strongly prevented by exogenous treatment with DRGN-1 (Fig. 2a–d). Exogenous application of DRGN-1, but not VK25, significantly reduced the number of internalized bacteria with the peptide causing 64% reduction in survival of the internalized bacteria at the concentration used (30 µg/ml) after 2 h (Fig. 2e). LL-37 treatment of infected HEKa cells also caused a significant reduction in internalized P. aeruginosa cells. Both of these reductions were statistically significant compared to untreated or VK-25-treated cells. We conclude that DRGN-1 treatment of P. aeruginosa-infected HEKa keratinocytes leads to increased killing of those bacteria, whether directly by the peptide (direct antibacterial effect) or by a peptide-induced response of the keratinocyte cells (host-directed effect).

Fig. 2 Treatment of cells with peptide reduces intracellular P. aeruginosa bacteria. a Light microscopy images of uninfected HEKa keratinocytes. b–d About 100,000 cells were infected with P. aeruginosa ATCC 9027 (1 × 107 CFU/ml) for 2 h, washed with PBS, treated with gentamicin to remove extracellular bacteria for 1 h, and b not-treated, or c treated with DRGN-1 (30 µg/ml), or (D) VK25 (30 µg/ml) for 2 h at 37 °C. e Bacterial survival within infected HEKa keratinocytes upon peptide treatment at 2 h post treatment. 100% represents the number of bacteria found inside of untreated HEKa cells. f Cytotoxicity testing: the peptides were tested for their cytotoxic effect against HEKa cells. The number of metabolically active cells remaining was determined by the MTT assay and is expressed as percentage with respect to the non-peptide-treated control cells. P values were calculated using a two-tailed t test (assuming unequal variances) comparing test strains to untreated control (*P < 0.05; **P < 0.01) Full size image

The cytotoxic effect of DRGN-1 and VK25 on HEKa cells was studied using an MTT (3-(4, 5-dimethylthiazolyl-2)- 2,5-diphenyltetrazolium bromide) based assay. As shown in Fig. 2f, both DRGN-1 and VK25 were devoid of toxic effects against these mammalian cells at a concentration range of 12.5–100 µg/ml. However, the control CAMP, LL-37, caused a significant decrease in HEKa cell viability at concentrations ranging from 50–100 µg/ml (Fig. 2f). DRGN-1 also did not show any toxicity against erythrocytes in a hemolysis assay at a high concentration (Fig. S2).

Effects of DRGN-1 on membrane permeability

Measuring the uptake of the fluorescent probe, ethidium bromide (EtBr), allowed us to assess the permeabilization of P. aeruginosa ATCC 9027 by these peptides. EtBr will only fluoresce if it is able to permeate both the inner and outer membranes through pores, and intercalate into the bacterial nucleic acid. The uptake of EtBr was significantly higher in the presence of DRGN-1 or LL-37 than in the presence of VK25 or the untreated control (Fig. 3a), suggesting significant disruption of the bacterial cytoplasmic membrane.

Fig. 3 Membrane permeability, and membrane potential. a Fluorescent dye uptake for EtBr by the P. aeruginosa ATCC 9027 in the presence of peptides (20 µg/ml). P values were calculated using a two-tailed t test (assuming unequal variances) comparing with untreated control at 20 min (**P < 0.01). b Membrane depolarization was monitored by the change in the intensity of fluorescence emission of the membrane-potential-sensitive dye DiSC 3 (5) (excitation wavelength at 622 nm, emission wavelength at 670 nm) on addition of 20 µg/ml peptides or 0.1% Triton X-100 for a positive control Full size image

The effect of CAMPs on the membrane potential was measured by monitoring the membrane potential using the fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide (DiSC 3 (5)) (Fig. 3b).20 DiSC 3 (5) dye is taken up by bacterial cell membranes in proportion to the electrical potential gradient across the bacterial cytoplasmic membrane. The dye concentrates in the cytoplasmic membrane, where it is able to self-quench its own fluorescence. Any compound that can permeabilize the cytoplasmic membrane and thus depolarize the gradient, such as an antimicrobial peptide, will lead to the release of DiSC 3 (5) and thus leads to an increase in fluorescence.21 The peptides were tested against P. aeruginosa ATCC 9027 at a concentration of 20 µg/ml, which was expected to cause a large and immediate increase in fluorescence (indicative of loss of membrane potential). DRGN-1 showed ~10% depolarization relative to Triton X-100 (which served as the “100%” positive control), while VK25 did not have any effect. These results suggest that in addition to membrane disruption, DRGN-1, much more than VK25, permeabilizes the membranes and slightly depolarizes the membrane potential.

Structure analysis of DRGN-1 by circular dichroism (CD) spectroscopy

The reversal of two N-terminal amino acids (ser–pro) in a histone H1-derived peptide of V. komodoensis caused drastic changes in antimicrobial activity, anti-biofilm activity, membrane permeability, and DNA binding. Therefore, we sought to determine the secondary structure of the peptides in the membrane-mimicking media, sodium dodecyl sulfate (SDS) and trifluoroethanol (TFE) (Fig. 4) using CD. Cathelicidin AMPs (such as LL-37) maintain a random or disordered structure until associated with a bacterial membrane or detergent micelle.22 The detergent SDS is able to form micelles that have a negatively charged surface,23 which mimics the bacterial membrane and forces the CAMP into a more ordered conformation such as a helix.24, 25 TFE is used in CD to promote a helical structure26 and stabilize secondary structure.27

Fig. 4 CD spectra of peptides in the presence of SDS or TFE. a, b CD spectra of 100 μg/ml concentration of peptides in the presence of the indicated concentrations of sodium phosphate buffer (PB), SDS or TFE. c Helical wheel plot of DRGN-1 predicted by HeliQuest. d Composition of α-helix conformation calculated from the ellipticity value at 222 nm using the relation (% α-helix = ([θ 222 ]−3000)/(−36000−3000)). The ratio [θ] 222 /[θ] 208 obtained by CD spectroscopy is defined as R2 value. R2 >1 is the hallmark of coiled-coil α-helical structure with >80% helical content (32) Full size image

By qualitatively evaluating the spectra, we conclude that DRGN-1 and VK25 showed a random coil conformation in aqueous solution (10 mM phosphate buffer) and 60 mM SDS solution. The α-helical content of DRGN-1 and VK25 were 22.3 and 32.8% in 50% TFE, respectively, with the percent contribution to secondary structure was measured using methods determined by Raussens et al.28 Both DRGN-1 and VK25 have a very low hydrophobic moment (0.013 and 0.088, respectively) compared with the highly α-helical LL-37 (0.521), suggesting that DRGN-1 and VK25 are not α-helical (Table 2). Nevertheless, DRGN-1 (0.64) displays a slightly larger R2 value, the hallmark of the coiled-coil α-helical structure in 50% TFE, than VK25 (0.44). It has been shown by several groups that TFE can induce and support α-helical structures in peptides.29, 30 These results suggest that the reversal of amino acids at the N-terminal contributes to an overall conformational change.

Table 2 Summary of CAMPs used in this study Full size table

Efficacy of DRGN-1 on in vivo wound healing

We have demonstrated that DRGN-1 peptide significantly inhibited biofilms of P. aeruginosa and S. aureus separately, and a mixed biofilm. Since biofilm may protect bacteria during infection, we sought to test whether application of DRGN-1 to an infected wound would promote clearance and healing.

To evaluate the impact of a mixed biofilm infection on an open wound, we developed a wound model using BALB/c mice. We have previously characterized P. aeruginosa and S. aureus-mixed biofilms for their heterogeneity and composition;19 these biofilms were generated and applied to a mouse wound. In our model, we will test whether wound healing in P. aeruginosa and S. aureus biofilm-infected skin wounds is significantly improved following peptide treatment compared to an untreated, infected wound, following the design of other published studies.31

We have previously characterized a mixed biofilm of P. aeruginosa and S. aureus,19 which is applied to the wound as the source of infection. Using this model, we evaluated the potential clinical application of DRGN-1 against P. aeruginosa/S. aureus biofilm-infected wounds (Fig. 5). Full-thickness round wounds of 6 mm in diameter were made between the shoulder blades of mice and overlaid with a mixed biofilm of P. aeruginosa/S. aureus grown on agar for 2 days.19 The kinetics of wound closure was evaluated by measuring the original wound area before and after treatment with peptides. DRGN-1, VK25, and LL-37 (20 μg/wound) were applied topically to their specified groups every other day until day 6. Reconstitution buffer was used as a control in this experiment. Treatment with DRGN-1 on a mixed biofilm-infected wound significantly reduced wound size compared to the controls as soon as day 4 post treatment. When the wound dressing was taken off on day 6, wound healing sped up in both treated and untreated conditions as observed at day 9.

Fig. 5 Efficacy in wound healing of peptides in a mixed biofilm-infected mouse skin wound model. a Schematic experimental design that includes the timing of infection, drug treatments, dressing off. b Effect of peptides on closure of P. aeruginosa/ S. aureus-infected wounds in mice. Wounds shown are representative of the group. Six-millimeter-diameter excisions were made on the back of mice. Each wound was infected with a mixed biofilm (P. aeruginosa/ S. aureus) grown on a polycarbonate membrane for 2 days. DRGN-1, VK25 or LL-37 (20 µg) was topically treated every 2 days in 1% hypromellose (n = 6 mice per group). Mice were lightly anesthetized with isofluorane and photographed on the indicated days following treatment. c, d Assessment of bacterial colonization in P. aeruginosa/ S. aureus-infected wounds. Wound tissue samples were harvested on day 6 after treatment, and the number of CFU/wound was counted in a selective medium for each species. The median value in each group is shown as a horizontal bar (n = 6; **p < 0.01 and ***P < 0.001) Full size image

Wounds treated with DRGN-1 were consistently smaller than wounds treated with phosphate buffered saline (PBS) buffer, VK25, or LL-37 (Fig. 5b, Fig. S3). The results showed that the difference in size was apparent by day 4 after the first treatment. By day 11, the wounds treated with DRGN-1 were completely healed, while the wounds treated with PBS, VK25, and LL-37 were not. After 6 days, the first treatment (three treatments), the bacterial counts of S. aureus and P. aeruginosa were significantly reduced in the DRGN-1-treated and LL-37-treated groups compared to the control group (Fig. 5c, d). In contrast, there was no significant reduction in S. aureus and only a slight reduction in P. aeruginosa in VK25-treated wound areas. These results indicate that DRGN-1 accelerates skin wound closure and healing, and reduction of bacterial counts in the wounds of P. aeruginosa/ S. aureus biofilm-infected mice. When the wound dressings were taken off on day 6, wound healing was accelerated in both treated and untreated conditions due to the air exposure, as observed at day 9 and 11 post treatment. In addition, hematoxylin–eosin staining of skin at day 11 clearly demonstrated that the skin layers were completely rehabilitated in the DRGN-1-treated wounds (Fig. S4).

To precisely dissect the effect of DRGN-1 on re-epithelialization and new tissue formation in wound, we then investigated the in vivo wound closure activity of DRGN-1 in a mouse full-thickness skin (uninfected) wound model. In this study, we used a silicone splint32 to avoid contraction of an extensive subcutaneous striated muscle layer, which is specific to mice, called the panniculus carnosus that is largely absent in humans.33 Full-thickness round wounds of 6 mm in diameter were made between the shoulder blades on mice, and the wound closure was evaluated by measurement of original wound area (%). When DRGN-1 or VK25 (20 μg/wound) was applied topically every 2 days post injury, DRGN-1-treated wounds were consistently smaller than wounds treated with PBS or VK25 (Fig. 6). At day 6, the wounds treated with DRGN-1 were smaller in area (41% compared to original wound area) than the control (65%) or VK25-treated (85%) wound. These results indicate that DRGN-1 peptide directly accelerates skin wound closure and healing in mice. In summary, wound healing was significantly enhanced by DRGN-1 in both uninfected and mixed biofilm (P. aeruginosa and S. aureus)-infected murine wounds.

Fig. 6 Wound healing of DRGN-1 in a mouse full-thickness skin (uninfected) wound model. a Schematic experimental design that includes the timing of drug treatments. b Effect of DRGN-1 on closure of full-thickness excisional wounds (6-mm-diameter) in mice. Each wound was treated with DRGN-1 or VK25 every 48 h (n = 5 mice per group). The same animal was lightly anesthetized and photographed on the indicated days following injury. c Changes in percentage of wound area at each time point in comparison to the original wound area. *P < 0.05 and **P < 0.01 Full size image

Efficacy of DRGN-1 on keratinocyte migration in a scratch-wound closure assay

Some CAMPs also have host-directed functions and thus CAMPs are also called host-defense peptides. One of the potential functions of CAMPs is the activation of keratinocyte migration resulting in the promotion of skin wound healing,9, 34, 35 which may contribute to the in vivo results we observed. Therefore, we tested the capacity of DRGN-1 to induce wound closure via this mechanism.

We first investigated whether CAMPs induced the migration of HEKa cells into a wound area using a scratch wound closure assay. The migration of cells into the wounded area was significantly increased in the presence of DRGN-1, as well as LL-37, compared to migration in the control within 7 and 24 h after wounding (Fig. 7a, b). There, significantly less wound closure activity was observed in the presence of VK25, which was slightly more than the control treatment (peptide reconstitution buffer). This suggests that part of the wound-healing benefit of DRGN-1 peptide is the promotion of keratinocyte migration. Subsequently, in order to determine whether the wound closure was influenced by an increased proliferation of keratinocytes upon their exposure to DRGN-1, the wound healing assay was carried out in the presence of 20 μM mitomycin C to block cell proliferation. As shown in Fig. 7c, d, mitomycin C strongly inhibited the migratory activity of keratinocytes induced by DRGN-1 (10 μg/ml). This suggests that the proliferation of HEKa cells highly contributes to the wound-healing effect produced by DRGN-1. To further understand the pathway by which DRGN-1 activates keratinocyte migration and proliferation, epidermal growth factor receptor (EGFR) was selected as a potentially activated kinase, because it has been reported to be central to the regulation of keratinocyte migration and proliferation.35,36,37 Previous studies have shown that LL-37 peptide can induce transactivation of the EGFR.35, 38

Fig. 7 'Scratch' assay for HEKa cells. a Confluent cells in medium with HGKS were wounded by a scratch with a pipette tip, and ‘gap’ closure in the presence of peptides (10 µg/ml) was monitored by light microscopy at 0, 7 and 24 h post peptide treatment. b Relative wound closure of separate experiments using either 1 or 10 µg/ml of peptide as indicated was calculated from micrographs using ImageJ software. The results shown are combined from three independent experiments; error bars represent the mean ± SD. The dotted lines indicate the control values for easier comparison to the other bars. c HEKa cells were pre-incubated with 20 µM mitomycin C for 90 min or 0.2 µM AG1478 for 10 min and subsequently treated or not with 10 μg/ml DRGN-1, as indicated. In parallel, cells treated with the peptide alone were included for comparison. Cells incubated with medium served as a control (Ctrl). d Relative wound closure. All data are the means of at least three independent experiments ± SE. The levels of statistical significance between groups are indicated as follows: *P < 0.05 Full size image

AG1478 is an inhibitor of EGFR tyrosine kinase, which blocks the activation of EGFR.38, 39 Interestingly, pretreatment of HEKa cells with 0.2 μM AG1478 prevented cell migration induced by DRGN-1. To examine potential EGFR pathway activation by DRGN-1, we investigated EGFR phosphorylation by Western blotting with an anti-phospho-EGFR antibody. DRGN-1 treatment led to phosphorylation of the EGFR at 5 min and the phosphorylation persisted for 20 min, which was not observed in AG1478-treated cells (Fig. 8a–c). The amount of EGFR protein did not change during this time period and treatment with human keratinocyte growth supplements (HKGS) containing 0.2 ng/ml of human epidermal growth factor (EGF) was used as a positive control. EGFR signaling pathway initiates several signal transduction cascades, such as STAT3 (Signal Transducer and Activator of Transcription) and Akt with the functional consequence of enhanced cell migration.40 STAT3 was also phosphorylated 10–30 min after the addition of DRGN-1 (Fig. 8d–f). From these results, we conclude that DRGN-1 induces keratinocyte migration via EGFR-STAT3 pathway activation.