Peptide Stability Is Important but not a General Requirement for Antimicrobial and Antibiofilm Activity in Vitro and in Vivo

Abstract

Peptide stability to proteases has been a major requirement for developing peptide therapeutics. This study investigates the effects of peptide stability on antimicrobial and antibiofilm activity under various conditions. For this purpose, two human cathelicidin-derived peptides differing in stability to proteases were utilized. While GF-17, a peptide derived from the major antimicrobial region of human LL-37, can be rapidly cleaved by proteases, the engineered peptide 17BIPHE2 is resistant to multiple proteases. In the standard antimicrobial susceptibility, killing kinetics and membrane permeabilization assays conducted in vitro using planktonic bacteria, these two peptides displayed similar potency. The two peptides were also similarly active against methicillin-resistant Staphylococcus aureus (MRSA) USA300 prior to biofilm formation. However, 17BIPHE2 was superior to GF-17 in disrupting preformed biofilms probably due to both enhanced stability and slightly higher DNA binding. In a wax moth model, 17BIPHE2 better protected insects from MRSA infection-caused death than GF-17, consistent with the slower degradation of 17BIPHE2 than GF-17. Here peptide antimicrobial activity was found to be critical for in vivo efficacy. When incorporated in the nanofiber/microneedle delivery device, GF-17 and 17BIPHE2 displayed a similar effect in eliminating MRSA in murine chronic wounds, underscoring the advantage of nanofibers in protecting the peptide from degradation. Since nano-formulation can ease the requirement of peptide stability, it opens the door to a direct use of natural peptides or their cocktails for antimicrobial treatment, accelerating the search of effective antibiofilm peptides to treat chronic wounds.

INTRODUCTION

Host defense antimicrobial peptides (AMPs) are important effector molecules in innate immunity for rapid elimination of invading pathogens.^1–4 Since AMPs are expressed as precursors, proteases play a critical role in regulating the release of mature peptides. In the case of the only human cathelicidin, the 18 kDa precursor protein (hCAP-18) can be cleaved in different manners, depending on their locations and proteases in the human body. In neutrophils, hCAP-18 is cleaved into LL-37 by proteinase 3.⁵ Subsequently, LL-37 folds into a long amphipathic helix (residues 2–31) for bacterial killing without the requirement of the disordered C-terminal tail (Figure 1A).⁶ A different peptide ALL-38, with one additional alanine at the N-terminus of LL-37, is identified in the human reproductive system.⁷ In this case under acidic conditions, female gastricsin is responsible for the cleavage of hCAP-18 from male sperm. In vitro, ALL-38 is as active as LL-37 in inhibiting bacterial growth. Under a diseased condition, the precursor is cleaved into TLN-58, a 58-residue peptide, including LL-37.⁸ In addition, LL-37 can also be cleaved into smaller fragments in human sweats or skin.⁹

Figure 1.

Backbone structures of (A) LL-37 (sequence: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), (B) GF-17 (sequence: GFKRIVQRIKDFLRNLV-amide), and (C) 17BIPHE2 (sequence: GBKRLVQRLKDBLRNLV-amide) determined by multidimensional NMR spectroscopy. GF-17 corresponds to the major antimicrobial region of LL-37 (residues 17–32, underlined) discovered by NMR-trim. 17BIPHE2 was engineered based on GF-17 by changing I20, I24, and L28 to D-leucines (underlined). In addition, two phenylalanines (F) were converted to biphenylalanine (B in the sequence) to enhance peptide activity against MRSA.

Some pathogens also secret proteases as a virulence factor to compromise the innate immune defense. LL-37 can be cleaved by aureolysin, a Staphylococcus aureus protease, at multiple sites, whereas the Staphylococcal V8 protease has a preferred cleavage site after an acidic residue.¹⁰ Streptococcus suis can evade the killing of LL-37 by up-regulating the ApdS protease, leading to LL-37 truncation. The N-terminally LLG cleaved peptide (LLGCP) is less bactericidal and less helical due to the disruption of the hydrophobic cluster between the N-terminal L1L2 and subsequent F5F6 of LL-37 in the membrane-bound 3D structure.⁶ Unlike LL-37, LLGCP is less chemotactic and inhibits the formation of neutrophil extracellular traps (NETs).¹¹ Uropathogenic Escherichia coli (UPEC) secretes OmpT to cut LL-37 into inactive fragments.¹² Pathogenic Candida albicans also releases 10 distinct secreted aspartic proteases to inactivate LL-37.¹³ Two intermediate fragments, LL-25 and SK-29, retain antifungal activity. While LL-29 also remains immune modulatory, LL-25 is unable to. It is found recently that group A Streptococcus (GAS) also cleaves LL-37 into two fragments: N-terminal LL-8 and C-terminal SK-29, leading to reduced chemotactic property but not antibacterial activity.¹⁴ Tannerella forsythia is a periodontal Gram-negative pathogen that can produce multiple proteases. Mirolysin, a 66-kDa protease, inactivates LL-37 and abolishes its binding to lipopolysaccharides (LPS).¹⁵ Hence, the combined action of host and pathogenic proteases during infection determines the molecular form and lifetime of human cathelicidin LL-37 as well as other host defense peptides (over 100 discovered in humans).⁴

Because of protease cleavage, an engineered stable peptide is preferred for therapeutic use so that it has a sufficient time to get rid of pathogens. To improve the stability of human LL-37 to proteases, D-amino acids, tryptophan, terminal amidation, and acetylation were utilized to increase the stability of EFK-17 to proteases.^16,17 Our identification of the minimal antimicrobial region (KR-12) of human LL-37 has stimulated interests in peptide engineering.^6,17 Recently, Göransson and his student synthesized a cyclic peptide based on KR-12.¹⁸ It is possible to obtain even shorter antimicrobial peptides based on KR-12 fragments in conjugation with fatty acids. A two-dimensional molecular array led to the identification of C10-KR8, which is highly selective and potent against methicillin-resistant Staphylococcus aureus (MRSA). When made in D-amino acids, this peptide gains additional advantages with both reduced serum protein binding and increased peptide stability to a variety of proteases.¹⁹ We also succeeded in engineering the major antimicrobial peptide GF-17 (residues 17–32) of human LL-37 (Figure 1B)²⁰ into a potent, stable, and selective form by introducing non-standard amino acids, including three D-amino acids and two biphenylalanines.²¹ This engineered peptide 17BIPHE2, with an essentially nonhelical structure (Figure 1C),²² gained activity against MRSA as well as other ESKAPE pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. In addition, 17BIPHE2 and its analogs are effective in a catheter-associated biofilm murine model^21,23 and a chronic wound healing model.²⁴ However, it is not yet demonstrated, especially in animal models, whether the engineered peptide 17BIPHE2 has any advantages over the natural fragment GF-17 in protecting animals from MRSA infection.

In this study, we first compared the effects of proteases and human serum on the stability of GF-17 and 17BIPHE2 in the presence of a single protease or a mixture of proteases from human serum. Our new results established that 17BIPHE2 could resist the action of trypsin, chymotrypsin and pepsin, whereas GF-17 was rapidly cleaved. Interestingly, these two peptides displayed similar antimicrobial activity against a panel of Staphylococcal clinical strains. They were also similar in killing kinetics and membrane permeation. These results set the stage for us to compare the antimicrobial effects of these two peptides against preformed biofilms in vitro as well as in vivo. Our results uncovered that GF-17 and 17BIPHE2 might, and might not, be similarly active depending on the type of experiments and peptide formulation.

MATERIALS AND METHODS

Bacterial Strains and Media.

The bacterial strains used in this study were Staphylococcus aureus USA300. In addition, multiple clinical strains were also utilized to compare the antimicrobial activity of GF-17 and 17BIPHE2. Tryptic soy broth (TSB) medium and Difco^™ Mueller Hilton Broth (MHB) medium for bacterial growth was obtained from BD Bioscience MD, USA.

Peptides.

The peptides used were synthesized chemically by Fmoc solid phase synthesis and purified on HPLC to purity >95% (GeneMed Synthesis, TX). Peptide quality was validated by mass spectrometry (MS), HPLC, and NMR spectroscopy. Peptide sequences were entirely consistent with NMR assignments.^20,22

Peptide Stability to Proteases Followed by HPLC.

Peptide stability was evaluated by incubating the peptide with a protease at 40:1 molar ratio at 37°C. Trypsin and chymotrypsin were purchased from Sigma and pepsin was obtained from Fischer Scientific, USA. Aliquots were taken for analysis on a reverse phase Waters HPLC system equipped with a C18 symmetry column (4.6 × 150 mm). The mobile phase started with 90% buffer A (water with 0.1% trifluoroacetic acid (TFA)) and ended at 100% buffer B (acetonitrile with 0.1% TFA) over a 20 min run. The peptide peak was detected by UV at 220 and 280 nm. Chemicals were obtained from Fishers.

Peptide Stability in Human Serum.

Peptide (~200 μM) was mixed with 25% human serum in dermal cell basal medium and incubated at 37°C. At a defined timepoint, the reactions were vortexed. Aliquots were then taken and precipitated with nine volumes of chilled ethanol for 20 min at −20°C. Precipitates were removed via centrifugation on a bench centrifuge at 16,100 g for 5 min. The supernatant was taken and then dried on a speedvac. The sample was resuspended in ultrapure water for HPLC analysis as described above.

Minimal Inhibitory Concentrations (MIC) of Peptides.

The assay was performed as described previously with minor modifications.²⁵ In short, the bacterial strains were inoculated overnight. These cultures were then freshly inoculated and allowed to reach the exponential growth phase. The cultures were diluted accordingly to ~10⁵ CFU/mL in TSB or MHB (different concentrations) and 90 μL of this solution was added to a 96- well microplate (Costar, Corning, NY) containing 10 μL of serially diluted peptide solutions (in water) and incubated overnight at 37ºC for 20 h. The growth as a function of absorbance was read with a CHROMATE microplate reader at 600 nm. The wells containing sterilized water instead of peptide served as the untreated control and the media was used as the blank.

Effects of Proteases on Peptide Activity.

Peptide antimicrobial activity was evaluated by incubating the peptide with a protease at 40:1 molar ratio for 1 h at 37°C. Post incubation, the peptides in the presence or absence of the protease was mixed with bacterial culture and incubated overnight in the same manner as described in the antibacterial assays.

Killing Kinetics.

Killing kinetics of S. aureus USA300 was conducted at a starting OD₆₀₀ 0.001 and treated with GF-17 or 17BIPHE2. Samples were taken from reactions at desired times: 15, 30, 60, 90, and 120 minutes and were diluted 400 times in 1×PBS buffer (Gibco, NY). Then, 50 μL was plated onto Mueller Hilton agar plates. The CFUs were determined after overnight incubation at 37°C.

Growth Inhibition.

Growth curves of S. aureus USA300 at a starting OD₆₀₀ of 0.1 per well in the absence and presence of different amounts of peptides (0.5×, 1×, and 2× MIC) were recorded in a FLUOstar Omega (BMG LABTECH, NC) microplate reader. The plate was incubated at 37°C with continuous shaking. Samples were read every 30 minutes for 48 cycles (24 h) with the absorbance wavelength set at 600 nm.

Membrane Permeabilization.

The peptide ladder was prepared as described for the antibacterial assay with 10 μL in a black COSTAR 96-well plate. Propidium iodide (PI) (MP Biomedicals, Solon, OH) was prepared in the dark and dissolved in DMSO (Thermo Fisher Scientific, NY) to 20 mM. This PI stock solution was further diluted to 1 mM with water and 2 μL of 1 mM PI was added to each well. Exponential phase MRSA USA300 was diluted to OD₆₀₀ = 0.11 with MHB and 88 μL was added to each well. The plate was incubated at 37°C with continuous shaking in a FLUOstar Omega (BMG LABTECH, NC) microplate reader. The sample was read every 5 minutes for 24 cycles with excitation and emission wavelengths of 584 nm and 620 nm, respectively.

DNA Retardation Experiment.

This experiment was conducted as described²¹ except for the use of the pUC19 plasmid.

Preformed Biofilm Disruption.

S. aureus USA300 was re-inoculated from the overnight culture to reach the exponential phase. A culture was made at ~10⁷ CFU/mL. 200 μL was then placed into each well of the 96-well microtiter plates. The plates were incubated at 37°C for 3, 6, 48, or 72 h to allow biofilm formation. Media was then pipetted out and the biofilms were washed with 1× PBS to remove the planktonic cells. 10× Peptide solution in 20 μL volume was added to 180 μL of fresh 10% TSB media. Plates were further incubated at 37°C for another 24 h. Quantification of the disruption of the biofilm by the peptide was done using XTT as described earlier.³⁵ Antibiofilm experiments using other bacteria (Escherichia coli, Staphylococcus epidermidis, and Pseudomonas aeruginosa) in supporting information followed the same procedure.

Invertebrate Wax Moth Model.

Wax moths (Galleria mellonella) in the larva stage (~250 mg) were distributed by Timberline Live Pet Foods Marion, IL. Peptide in phosphate buffer was injected into wax moths 2 h prior to the infection with S. aureus USA300 at 1×10⁶ CFU/insect. The insect groups (30 per group) included (1) PBS treated, (2) S. aureus USA300 infected without treatment, (3) LL-37 treated prior to S. aureus infection, (4) GF-17 treated prior to S. aureus infection, (5) 17BIPHE2 treated prior to S. aureus infection, and (6) RI-10 treated prior to S. aureus infection. LL-37 is the parent peptide of GF-17, whereas RI-10 is an LL-37 segment that has lost antibacterial activity. Animals were kept at room temperature and observed daily for 5 days to record live and dead changes.

Vertebrate Wound Healing Model.

We established a biofilm-containing chronic wound model following previous studies.^24,26 Briefly, MRSA USA300 was grown in LB broth medium overnight. Subsequently, 100 μL of bacterial strain was pipetted into 4 mL of fresh LB medium and cultivated for 3 h followed by PBS washing for three times. Then, the bacterial concentration was adjusted to ~1×10⁸ CFU/mL and stored in the ice box before use. Nine 000697-B6.BKS(D)-Leprdb/J diabetic defective mice (female, 8–9 weeks, 40–45 g, GLU > 200 mg/dL) fed with standard pellet diet and water were used. The animal study was approved by the Institutional Animal Care and Use Committee of University of Nebraska Medical Center (protocol #19–069-07-FC).

The biofilm was established in the 000697-B6.BKS(D)-Leprdb/J mouse excisional wounds. Two 6 mm-diameter full-thickness wounds were created on the back of a mouse using a disposable biopsy punch (Integra Miltex, Kai Medical) and fixed with splints. The wounds were inoculated with 10 μL of 1×10⁸ CFU/mL MRSA instantly after surgery, and 2% mupirocin was applied to treat the wounds at day 2 (24 h after surgery). After the establishment of biofilm-containing wound model, Janus-type dressings loaded with 25 mg/g D106 and 17BIPHE2 antimicrobial peptides were placed on the wounds, respectively, to treat the biofilms. Janus-type dressings consisting of electrospun nanofiber membranes and microneedle arrays were fabricated following our previously established protocols.^26,27 The wound and surrounding tissue was collected by a 10 mm-diameter punch into sterilized tubes after 48 h. Then, 1 mL of sterilized PBS was added to each tube, which was blended by a homogenizer. Subsequently, the mixed liquid was diluted and plated on agar dishes. All the dishes were incubated in a microbial incubator at 37°C for 18 h, and then the CFU were counted.

Statistical Analysis.

All the quantitative data are represented as mean ± standard deviation. The obtained data were analyzed for statistical significance using log-rank tests in the insect survival experiments and one-way ANOVA tests in the mouse wound healing model. In both cases, *p < 0.05 was considered statistically significant.

RESULTS

The Engineered Peptide 17BIPHE2 Is More Stable Than GF-17, a Synthetic Fragment of LL-37.

We compared the degradation kinetics of GF-17 and 17BIPHE2 (B) in the presence of either chymotrypsin or trypsin. The GF-17 peptide was rapidly degraded by chymotrypsin with less than 50% left after 1 h (t_1/2 < 60 min, Figure 2A). In contrast, there was little change to the peak height of 17BIPHE2 after two hours incubation (Figure 2B). Trypsin appeared to be more effective than chymotrypsin in cleaving GF-17 as the peptide peak was essentially lost after 67 or 120 min incubation (t_1/2 < 60 min in Figure 2C). However, we only observed a minor decrease in peak height of 17BIPHE2 at 2 h compared to that at zero min (Figure 2D). Evidently, 17BIPHE2 was more resistant to protease degradation than GF-17. To validate these results, growth inhibitory experiments were conducted. Different from normal minimal inhibitory concentration (MIC) experiments, the serially diluted peptides were first incubated with a protease (e.g., chymotrypsin, trypsin, or pepsin) for 30 min. In all the three cases, GF-17 in the range of 2–32 μM did not inhibit the growth of S. aureus USA300 after overnight incubation (Figure 3), but 17BIPHE2 did show an inhibitory effect at 4 μM in the case of chymotrypsin (Figure 3A) or pepsin (Figure 3C) and at 8 μM in the presence of trypsin (Figure 3B). All these experiments indicate that 17BIPHE2 could kill MRSA even in the presence of these proteases.

Figure 2.

Peptide stability to proteases followed by HPLC: (A) GF-17 + chymotrypsin; (B) 17BIPHE2 + chymotrypsin; (A) GF-17 + trypsin; (B) 17BIPHE2 + trypsin. The peptide:protease molar ratio was 40:1.

Figure 3.

Growth inhibition of Staphylococcus aureus USA300 LAC with and without protease pretreatment: (A) chymotrypsin; (B) chymotrypsin; and (C) pepsin. The procedure is the same as MIC assays except for protease pretreatment.

We then also compared the stability of the peptides in human serum. While GF-17 was essentially degraded after 24 h incubation (Figure 4A–D), there was little loss in the peak height of 17BIPHE2 after 24 h digestion (Figure 4E–H). Therefore, 17BIPHE2 was also more stable in human serum. Taken together, these experiments established that 17BIPHE2 is more stable than GF-17 to protease degradation by either a single pure protease or a mixture of proteases in human serum.

Figure 4.

Degradation kinetics of GF-17 (left) and 17BIPHE2 (right) in 25% human serum. Samples were taken after incubation at 37°C for 0, 2, 5, and 24 h.

In Vitro Antimicrobial Susceptibility against a Panel of Planktonic S. aureus Clinical Strains.

Human LL-37 was not active in 100% TSB or MHB against S. aureus USA300 (MIC >32 μM in Table 1) although it gained activity in the diluted media. In contrast, the anti-Staphylococcal activity of both GF-17 and 17BIPHE2 was similar and essentially not influenced by the concentration change of MHB (Table 1). Moreover, both peptides retained similar activity at pH 6.8, 7.4, and 8.0. GF-17 and 17BIPHE2 also showed the same MIC value in the presence of 150 mM NaCl. However, human serum reduced the activity of the peptide similarly at 5% serum (Table 2). At 10% 17BIPHE2 retained activity, while GF-17 became inactive. No difference could be observed in the presence of 20% serum.

Table 1.

Effects of Media and MHB concentrations on LL-37 peptide activity (MIC, μM) against methicillin-resistant Staphylococcus aureus USA300 LAC

Medium	LL-37	GF-17	17BIPHE2
100% TSB	>32	4	4
100% MHB	>32	2–4	4
50% MHB	32	2	2
25% MHB	8–16	2	2
12.5% MHB	4–8	2	2
6.25% MHB	4	2	2

Table 2.

Effects of salt, human serum and pH on minimum inhibitory concentration (μM) of GF-17 and 17BIPHE2 ¹

Peptide	pH 6.8	pH 7.4	pH 8.0	150 mM NaCl	5% Serum	10% Serum	20% Serum	Chymo	Trypsin	Pepsin
GF-17	4–8	4	2	2–4	16	>32	>32	>32	>32	>32
17BIPHE2	4	4	2–4	2–4	16	8–16	>32	4–8	8–16	4–8

¹The parent molecule LL-37 was inactive in rich media against MRSA under all the conditions.

To further compare antibacterial susceptibility of 17BIPHE2 and GF-17, we also tested a panel of 31 clinical strains of S. aureus, including 5 methicillin-susceptible and 26 resistant strains (Table S1). They showed similar MIC values against all the strains. As a negative control, RI-10, a KR-12 truncated peptide, did not show any inhibitory activity at 32 μM. In these antimicrobial experiments (~10⁵ CFU/mL), peptide stability did not appear to play a role.

We also compared killing kinetics of 17BIPHE2 and GF-17. These two peptides were equally potent and eliminated S. aureus USA300 in less than 50 min when treated at 2× MIC (Figure 5A). Likewise, GF-17 and 17BIPHE2 showed similar membrane permeabilization at 0.5, 1, 2, and 4 fold of MIC, although GF-17 was slightly more effective than 17BIPHE2 at 0.25 MIC (Figures 5B & S1). As negative controls, non-membrane targeting antibiotics, nafcillin and linezolid, were unable to permeabilize bacterial membranes under the same conditions since the curves resemble the untreated S. aureus USA300 (Figure 5B). Membrane permeabilization enabled the entry of the fluorescent dye. In the same manner, the peptide might also enter bacteria and bind DNA as well. To confirm this, gel retardation experiments were conducted using the pUC19 plasmid (Figure S2). While there was a clear peptide dose-dependent DNA retardation in the well for both LL-37 and GF-17, 17BIPHE2 was sufficient to retard the DNA at 3 μM. As a control, RI-10 was unable to retard DNA at both peptide concentrations.

Figure 5.

S. aureus USA300 killing kinetics (A) and membrane permeabilization (B) by GF-17 and 17BIPHE2.

Next, we conducted a 24-h growth experiment in the presence of GF-17 or 17BIPHE2 at a starting OD₆₀₀ = 0.1 (~10⁷ CFU/ml, Figure 6). Without antimicrobial treatment, S. aureus reached an OD₆₀₀ ~1.5 in 24 h. When treated at 2× MIC, bacterial growth was completely suppressed by both peptides. At 0.5× MIC, S. aureus started to grow after a ~3 h delay. When treated at 1× MIC, additional delay was required for S. aureus to grow. The growth resumed after ~10 h when treated with GF-17. However, MRSA did not grow after ~15 h in the presence of 17BIPHE2 (Figure 6). This experiment revealed that 17BIPHE2 was more effective in preventing the growth of S. aureus USA300 than GF-17.

Figure 6.

Growth inhibition of the S. aureus culture treated at different fold of the minimal inhibitory concentration (MIC) of GF-17 or 17BIPHE2.

Effects of GF-17 and 17BIPHE2 on Bacterial Biofilms.

We then compared the antibiofilm activity of GF-17 and 17BIPHE2. Previously, LL-37 was found to be able to inhibit biofilm formation at low concentrations^28,29 but was ineffective against preformed biofilms of S. aureus.²⁸ RI-10, which was unable to kill bacteria,⁶ could not disrupt preformed biofilms, either. We then compared the antibiofilm activity of GF-17 (Figure 7 A–D) and 17BIPHE2 (Figure 7 E–H) by forming biofilms for different times. After 3 h (i.e., before biofilm formation), GF-17 and 17BIPHE2 eliminated essentially all S. aureus at 1× MIC. When six hours were allowed for biofilm formation, we started to see differences in bacterial killing. In the case of GF-17, S. aureus reduced from ~90% to ~40% treated at 1× and 2× MIC, respectively (Figure 7B). In contrast, only 25% S. aureus was alive at 1× MIC and no bacteria survived at 2 to 8× MIC (Figure 7F). Likewise in the 48 h preformed biofilms, 17BIPHE2 was more effective than GF-17. About 50% S. aureus remained at 4× MIC of GF-17, while the same amount was left at 1× MIC when treated with 17BIPHE2. Similar results were obtained for the 72 h biofilms where 17BIPHE2 was more disruptive.

Figure 7.

Disruptive effects of GF-17 (left) and 17BIPHE2 (right) on the biofilms of S. aureus USA300 LAC preformed for 3, 6, 48, and 72 h.

To exclude the possibility that this effect was solely due to MRSA, we also compared antibiofilm effects of GF-17 and 17BIPHE2 against the biofilms of E. coli, S. epidermidis, and P. aeruginosa. In all the cases, 17BIPHE2 remained more potent against the 48-h biofilms than GF-17, although the difference was smaller in the case of P. aeruginosa isolated from wounds (Figure S3).

Antimicrobial Efficacy in an Invertebrate Model.

Wax moths (Galleria mellonella) are a widely used model.^30,31 Using this model, we previously observed in vivo efficacy of WW298 as well as merecidin, a derivative of 17BIPHE2.^32,33 Without infection, we found no animal death up to 512 mg/kg of 17BIPHE2. After infection, log-rank tests revealed that the differences between all the treated groups and the uninfected group (PBS treated) were statistically significant (p < 0.013 to 0.001). As a positive control, LL-37 (64 mg/kg) showed some protective effect at day 5 (37% survived out of 30 animals per group). While GF-17 (63% survival) was better than LL-37, 17BIPHE2 was best (83% survival) in protecting larvae from infection-caused deaths (Figure 8). Notably, RI-10, which is unable to kill MRSA, did not protect the animals, similar to the untreated group. To provide insight into this activity difference, we also followed pharmacokinetics of GF-17 and 17BIPHE2 in insect hemolymph (Figure S4). The remaining peptide peaks for 17BIPHE2 after 10 min to 3 h incubation were clearly higher than those of GF-17. We concluded that the higher potency of 17BIPHE2 was correlated with its higher stability.

Figure 8.

Comparison of GF-17 and 17BIPHE2 efficacy in the wax moth model. For comparison, we included their parent peptide LL-37 and an LL-37 derived inactive peptide RI-10 (sequence: RIVQRIKDFL). Insects were separated into different groups (n=30). Peptide was injected at 64 mg/kg and the larvae were incubated at room temperature. Two hours post-infection, each insect was infected with 10⁶ CFU of S. aureus USA300 LAC. The difference between treated and the PBS treated groups is statistically significant (see Methods).

Antimicrobial Efficacy of GF-17 and 17BIPHE2 in a Wound Model.

Previously, we demonstrated the efficacy of 17BIPHE2 in treating chronic wound.²⁴ However, it was unclear whether 17BIPHE2 is better than GF-17 in this biofilm model in vivo. Here we compared the anti-biofilm efficacy of GF-17 and 17BIPHE2 in the same model. Briefly, wounds were created and fixed with a splint (Figure S5). 10 μL of ~10⁸ CFU/mL MRSA was inoculated to each wound to allow biofilm establishment for 24 h and followed by 24 h treatment with 2% mupirocin ointment to remove the planktonic bacteria in the wounds. For in vivo anti-biofilm testing, we applied Janus-type dressings loaded with GF-17 or 17BIPHE2 to the biofilm-containing wounds, respectively. After treatment for 48 h, the collected tissues from the wounds treated by the Janus-type dressings without loading any peptides contained 1.82×10¹⁰ CFU/g of bacteria, whereas only 2.82×10⁴ CFU/g and 1.44×10⁴ CFU/g of S. aureus USA300 LAC were detected in the wounds treated with Janus-type dressings loaded with GF-17 and 17BIPHE2, respectively. The treatment with the dressings containing either GF-17 or 17BIPHE2 peptide resulted in 6-Log reduction of bacterial burden (CFU/g, Figure 9). This result indicated that GF-17 and 17BIPHE2 peptide-loaded Janus-type antimicrobial dressings were equally potent against MRSA biofilms in chronic wounds.

Figure 9.

The anti-biofilm efficacy of Janus-type antimicrobial dressings in vivo. The dressings were placed on the wounds for 48 h. (*p < 0.05) PCL-F127+PVP MN: PCL-F127 nanofibers + PVP microneedle arrays without peptide loading. PCL-F127/GF-17+PVP/GF-17 MN: Janus-type dressing composed of GF-17 peptide-loaded PCL-F127 nanofibers and GF-17 peptide-loaded microneedle arrays. PCL-F127/17BIPHE2+PVP/17BIPHE2 MN: Janus-type dressing composed of 17BIPHE2 peptide-loaded PCL-F127 nanofiber membrane and 17BIPHE2 peptide-loaded microneedle arrays.

DISCUSSION

Human cathelicidin LL-37 has been widely investigated for its therapeutic potential.^17,34–36 A variety of LL-37 peptides have been designed to improve antimicrobial potency, peptide selectivity, and stability (Reviewed in ref.¹⁷). More recently, SK-24 was designed based on the 3D structure of LL-37. The SK-24 peptide appears to have stronger antibiofilm activity than GF-17 in the case of 72 h biofilms.³⁷ Also, even shorter lipopeptides with merely eight amino acids were identified via a peptide library screen. C10-KR8d, made in D-amino acids, is highly selective and potent.¹⁹ White et al. reported that an improved form of the cyclic KR-12 peptide shows promising results as a new antibiofilm agent.³⁸ In all these studies, peptide engineering to enhance peptide stability was an important part of the research. How peptide stability might influence the experimental outcomes is not well understood. Our current study has improved our understanding by evaluating the impact of peptide stability on antimicrobial activity under various conditions using GF-17 and 17BIPHE2 as model peptides. Multiple lines of evidence support that 17BIPHE2 (an engineered GF-17 peptide) is indeed more stable than GF-17 (a natural fragment of LL-37) to the action of chymotrypsin, trypsin, and pepsin under in vitro conditions as well as in human serum (Figures 2–4). Moreover, 17BIPHE2 is recently found to be more stable than GF-17 in cervicovaginal fluids.³⁹ However, peptide stability did not show a clear advantage in numerous in vitro experiments, including antimicrobial susceptibility assays (Tables 1–2 and Table S1), killing kinetics, and membrane permeabilization studies (Figure 5). These two peptides also displayed similar hemolysis with comparable 50% hemolytic concentrations (HC₅₀).³⁷ Taken together, these results underscore comparable antimicrobial and hemolytic ability of GF-17 and 17BIPHE2, making them an ideal peptide pair for us to compare their antibiofilm capabilities in vitro and in vivo as well. Our study underscores that this requirement is determined by both the peptide concentration and the initial CFU in the culture. During MIC assays, the peptide is sufficient to eliminate the bacteria, leading to the same activity for both GF-17 and 17BIPHE2. In the growth recovery experiment (Figure 6), again we do not see any differences when there are enough peptides to kill MRSA. When the bacteria were not all killed, the remaining could secrete protease to try to survive. This provides one possible reason for the recovery of the bacterial growth with time. The delay is longer in the case of the engineered peptides since it takes longer time to cleave it (Figure 6). Our results also indicate that the stability requirement also depends on the biofilm-forming time. It is known that bacteria are capable of forming biofilms in hours (6–8 h).^25,40 In the absence of biofilms (biofilm-forming time 3 h), we observed similar killing effects for GF-17 and 17BIPHE2. However, we observed consistently a better antibiofilm effect for 17BIPHE2 against MRSA biofilms formed for 6 h or longer (48–72 h). After 6 h growth, S. aureus has transformed to the exodus stage of the biofilms coated with extracellular DNA (eDNA).⁴⁰ This exodus includes secretion of proteases. Our unpublished results uncover the upregulation of the aureolysin gene of MRSA when treated at a sublethal level of cationic peptides for 30 min. Consistent with this observation, peptide stability is critical for anti-Ebola viral effects of 17BIPHE2 in protease-rich endosomes where GF-17 did not work in the same experiment.⁴¹ Since the biofilms of MRSA are coated with eDNA,⁴² it might be logical to assume that the peptide would initially interact with the bacterial eDNA outside of biofilms. Our DNA gel retardation experiment implied that 17BIPHE2 was more effective in retarding the DNA than GF-17 (Figure S2). This observation might be translated to more potent disruption of the outer DNA layer of the MRSA biofilms by 17BIPHE2 than by GF-17, enabling the rest peptide to eliminate pathogens initially hidden within biofilms via membrane targeting (Figure 5B).

We also compared the antimicrobial efficacy of these two LL-37 peptides in vivo. In the invertebrate model, 17BIPHE2 better protected wax moths than GF-17. One possible reason is the higher stability of 17BIPHE2 than GF-17 in agreement with peptide pharmacokinetics in insects (Figure S4). Note that RI-10, which is not antimicrobial, did not protect the animals for MRSA infection in the same experiment (Figure 8). Likewise, we observed previously in vivo efficacy for 17BIPHE2 in protecting the catheter from MRSA infection in a murine model, whereas LL-23V9,⁴³ which was not active against MRSA USA300, did not show any decrease in bacterial burden in vivo.²¹ These results underscore that peptide antibacterial activity is critical for the observed in vivo efficacy either in insects or in mice.

Proteases might also exist in chronic wounds where bacterial biofilms have established. It was speculated that the lack of stability might be the reason why a direct treatment with free peptides such as LL-37 was ineffective.⁴⁴ However, we observed similar treatment outcomes for GF-17 and 17BIPHE2 in a chronic wound model (Figure 9). This is because the peptides are incorporated in nanofibers, which restricted the access of proteases to peptides in vivo. When released, both peptides can rapidly kill pathogens, leading to similar in vivo efficacy in this model. This observation implies the direct use of non-engineered synthetic peptides for antibiofilm treatment. Indeed, W379 (i.e., verine),⁴⁵ a short peptide made with L-amino acids, was as effective as 17BIPHE2/nanofiber in the same model after formulation.²⁶ This practice would remove the cost of peptide engineering. The reduced cost from peptide engineering would compensate for the cost from nanofibers, making the technology more affordable.

The picture is more complex in vivo. Although not the focus of this study, another important factor also in play is the ability of peptides to associate with various molecules in vivo. Using the mass spectrometry-based proteomic approach, we previously identified numerous serum proteins that could bind to LL-37 derived lipopeptides obtained from a 2D molecular array. Such a binding appears to depend on the peptide type. The peptide with a native sequence preferentially associated with many more serum proteins. In contrast, the peptide made using non-standard D-amino acids (D-form) showed substantially reduced binding.¹⁹ Hence, the D-form peptides offer one extra advantage of reduced serum protein binding in addition to enhanced stability to proteases. We noticed here that GF-17, with a regular helical structure (Figure 1B),²⁰ tends to bind more serum proteins than 17BIPHE2 with an irregular backbone structure (Figure 1C).²² We estimated the bound portions based on the HPLC samples with and without serum. In the case of GF-17, 66.7% were lost due to a short time mixing with serum. In contrast, 45.6% of 17BIPHE2 were bound to human serum. The reduced serum protein binding of 17BIPHE2 than GF-17 may be attributed to the difference in backbone structure. The helical structure of GF-17 enabled more protein binding than the non-helical structure of 17BIPHE2 (Figure 1). This molecular binding is a rapid physical process, while the protease cleavage in serum is a slower chemical process as we followed in Figures 4 and 5. Hence, only a portion of free peptides will be available to direct bacterial killing in vivo. We propose that serum protein binding is also minimized when the peptides are formulated in nanofibers for controlled release.

CONCLUSIONS

With the shift toward precise medicine, we anticipate increased importance of peptide therapeutics. The classic wisdom is that peptide stability is critical for potential therapeutic utilization. Consequently, molecular engineering becomes a major effort to enhance peptide stability to host and pathogen proteases. This article investigated the impact of peptide stability on antimicrobial and antibiofilm activity under a variety of in vitro and in vivo conditions using a pair of human LL-37 derived peptides as models. In numerous planktonic cases, GF-17 and 17BIPHE2 are equivalent in inhibiting MRSA in antimicrobial susceptibility, killing kinetics, and membrane permeation experiments. It is not essential, either, before biofilm matures. However, 17BIPHE2 did show advantages in disrupting MRSA biofilms. We propose that both peptide stability and DNA-binding capability play a role. In the invertebrate model where the peptide is fully exposed to the protease-containing biological matrix, peptide stability is clearly important for in vivo efficacy. After formulation for controlled release, however, peptide stability becomes less important in killing MRSA in biofilms. These results are useful for choosing a right peptide form (free and formulated) depending on the experimental scenarios. The use of non-engineered (i.e., protease-susceptible) peptides in nanofibers for controlled release can not only compensate for the cost of peptide formulation but also speed up the search of optimized peptides and their cocktails for optimal wound healing. Peptide cocktails, more resembling the natural states, may enhance killing power due to synergistic effects, further reducing the likelihood of the development of bacterial resistance^46–48 to these membrane-targeting host defense peptides.^49–51

Supplementary Material

ASSOCIATED CONTENT

Supporting information is available.