Enhanced Antimicrobial Screening Sensitivity Enabled the Identification of an Ultrashort Peptide KR-8 for Engineering of LL-37mini to Combat Drug-resistant Pathogens

Abstract

Identification of novel antibiotics is of top importance because of the threat of antibiotic-resistant pathogens. Antimicrobial screening in Mueller Hinton broth is frequently the first step in antimicrobial discovery. Although widely utilized, this medium is not ideal as it could mask activity of candidates such as human cathelicidin LL-37 against methicillin-resistant Staphylococcus aureus (MRSA). This study identified a sensitive medium where LL-37 displayed excellent activity against numerous pathogens, including MRSA. Our screen of ultrashort overlapping LL-37 peptides in this medium led to the identification of KR-8, four-residue shorter than KR-12. Hence, our screen condition may increase positive compound hits during antimicrobial screening. KR-8 provided an appealing template for us to design LL-37mini, which was potent against MRSA, Escherichia coli, and Pseudomonas aeruginosa but was not toxic. LL-37mini also inhibited bacterial attachment, biofilm formation, and disrupted preformed biofilms in vitro and killed MRSA in murine wound biofilms in vivo. Consistent with membrane targeting, MRSA failed to develop resistance to LL-37mini in a multiple passage experiment. Because LL-37mini can be made cost effectively, it can be developed into new antibiofilm and antimicrobial agents.

A British report projected the deaths of 10 million people by 2050.¹ Because of lasting potency, antimicrobial peptides (AMPs) are important candidates for developing future antibiotics.^2–6 According to the antimicrobial peptide database (APD; https://aps.unmc.edu), over 3000 AMPs have been isolated and characterized from six life kingdoms, including bacteria, archaea, protists, fungi, plants, and animals.^7,8 Like plants and insects, humans also deploy multiple antimicrobial peptides (AMPs) in response to a variety of invading pathogens, including bacteria, fungi, viruses, and parasites. While there are numerous defensins in humans,^2,9 a single cathelicidin gene is mapped in the human genome.^5,10 Different from human α and β-defensins, which comprise three pairs of disulfide bonds, human cathelicidin LL-37 does not contain cysteine and belongs to the linear peptide class. LL-37 is the most widely investigated form of human cathelicidin peptides with different lengths.

Earlier studies learned that the structure and activity of LL-37 depends on environmental conditions.¹⁰ In standard Mueller Hinton Broth (MHB) medium, Lehrer and colleagues observed antibacterial activity for LL-37 against Gram-negative pathogens, but not Gram-positive pathogens, such as Staphylococcus aureus.¹¹ They hypothesized that acidic components in the medium was responsible for masking the peptide activity. Indeed, they were able to show the bactericidal activity of LL-37 in a refined MHB. This refined medium was obtained by passing through a cationic column to remove the anionic components that had compromised peptide activity. Due to inconvenience, however, this refined medium has not been widely adopted for antimicrobial susceptibility assays. Hence, an easily accessible and cost-effective medium is required for antimicrobial susceptibility assays of LL-37 and other antimicrobial agents.

This study intended to identify a convenient screen condition to increase the positive hits of antimicrobial screen. Using this medium, we then screened a small library of ultrashort peptides (≤ 10 amino acids) covering the entire LL-37 sequence. Our screen identified an eight-residue fragment active against Escherichia coli. Finally, we demonstrated the design of potent peptides based on the newly identified minimal antimicrobial region of LL-37. Here we report these results.

RESULTS

Identification of a Medium Condition to Reveal the Antibacterial Activity of LL-37 against S. aureus USA300.

To identify a useful medium condition, we first compared antibacterial activity of LL-37 in several frequently used rich media: MHB, Tryptic Soy Broth (TSB), and Luria-Bertani (LB). These rich media were made as recommended and defined as 100% in this study. As anticipated, we failed to observe the inhibition of methicillin-resistant Staphylococcus aureus (MRSA) USA300 LAC by LL-37 in all these media until 32 μM (Table 1). As a positive control, we did observe bacterial killing for 17BIPHE2, a peptide engineered based on the core major antimicrobial peptide FK-16 of human LL-37 (amino acid sequences in Figure 1) by changing I20, I24, and L28 into D-leucine and F17 and F27 to biphenylalanine.¹²

Table 1.

Antimicrobial Activity (μM) of LL-37 and 17BIPHE2 in Different Media

		S. aureus USA300		E. coli E423–17
Media		LL-37	17BIPHE2	LL-37	17BIPHE2
MHB	100%	> 32	4	8	2–4
	50%	32	4	4–8	2
	25%	8	4	4	4
	12.5%	4	≤2	2–4	2–4
TSB	100%	>32	2–4	8	4–8
	50%	>32	≤2	4–8	4
	25%	>32	≤2	4	2
	12.5%	>32	≤2	≤2	≤2
LB	100%	>32	≤2	4	2–4
	50%	>32	≤2	4–8	2–4
	25%	>32	4	4	2
	12.5%	>32	4	≤2	≤2

Figure 1.

The sequence relationship of human cathelicidin LL-37 with its antimicrobial fragments LL-31, SK-24, FK-16, KR-12, and KR-8. The 3D structure of LL-37 (PDB: 2K6O) bound to membrane-mimetic lipids was determined by 3D triple-resonance NMR spectroscopy.¹⁵ KR-8, the template for designing LL-37min, is the smallest active peptide from LL-37 newly discovered in a sensitive medium. See the text for additional details.

We then tested anti-MRSA activity of LL-37 in diluted media. Interestingly, LL-37 inhibited the growth of MRSA with the dilution of MHB, but not the dilution of TSB nor LB (Table 1). As a positive control, the minimal inhibitory concentration (MIC) of 17BIPHE2 was barely compromised from 100% to 12.5% (2–4 μM). Importantly, the MIC (4 μM) value for LL-37 in 12.5% MHB was comparable to those of 17BIPHE2. Further antimicrobial assays revealed that such an enhanced activity in diluted media was also observed for Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii (Table 2). Notably, we found that media dilution had only a subtle effect on antibacterial activity of LL-37 against Escherichia coli (Table 1) and A. baumannii (Table 2). In all the cases, LL-37 displayed an optimal activity in 12.5% MHB. Remarkably, 17BIPHE2 displayed consistent MIC values under all the conditions tested (media, dilution, and bacteria), indicative of its antimicrobial robustness (Tables 1 and 2).

Table 2.

Media Dilution Effect on Antimicrobial Activity (μM) of LL-37 and 17BIPHE2 against Other Bacteria

Media	S. epidermidis 1457		P. aeruginosa 152		K. pneumoniae E406–17		A. baumannii B28–16
MHB	LL-37	17BIPHE2	LL-37	17BIPHE2	LL-37	17BIPHE2	LL-37	17BIPHE2
100%	>32	4	32	4	16–32	2–4	8	≤2
10%	2–4	4	4	≤2	4	4	4	4

LL-37 is known to inhibit bacterial biofilms.³ However, it was unable to disrupt preformed MRSA biofilms in rich media.¹³ Next, we asked whether the diluted MHB medium could be useful to detect biofilm-disrupting activity for LL-37 against MRSA. We followed an established procedure.^13,14 In this procedure, biofilms were formed and washed as usual to remove planktonic cells. The biofilms were then treated with peptides in 12.5% MHB for 24 h. Finally, live cells were quantified with XTT (ATCC). Figure 2 compares the levels of live cells of MRSA in the preformed biofilms after 24 h treatment. In 100% MHB, LL-37 showed essentially no antibiofilm activity, while 17BIPHE2 started to work at 8 μM and only ~30% live bacteria remained in the biofilms when treated at 16 μM. Of note is a similar pattern for both peptides in 12.5% MHB treated at various concentrations. LL-37 worked at 32 μM, while 17BIPHE2 showed clear biofilm disruption at 16 μM. Hence, we observed similar antibiofilm activity for LL-37 and 17BIPHE2 in this medium as well.

Figure 2.

Biofilm disruption activity of human LL-37 and 17BIPHE2 against the 24 h preformed biofilms of S. aureus USA300 LAC in different MHB media.

We then compared the antibiofilm activity of LL-37 and 17BIPHE2 against a clinical E. coli strain E423–17. In 100% MHB, both LL-37 and 17BIPHE2 showed a subtle effect on biofilms with the increase in peptide concentration. However, a dose-dependent antibiofilm effect is clear in 12.5% MHB (Figure S1). These results indicate that the 12.5% MHB (used only during treatment) is also useful for antibiofilm assays to avoid activity masking. This media dilution benefit appears to be media-dependent as we did not observe a similar trend for LL-37 when we diluted TSB and LB rich media in the same manner (Table 1).

We also compared the effects of MHB dilution on antimicrobial activity of a panel of antibiotics (Figure S2). For nafcillin and amikacin, we observed small changes in MIC values with the dilution of MHB from 100% to 12.5%, implying an effect of media on their activity. However, there were essentially no changes in antibacterial activity for daptomycin, gentamicin, linezolid, muporicin, tobramycin, and vancomycin with the dilution of MHB in the same manner (Figure S2). Since the majority of antibiotics (75%) showed similar MIC values with MHB dilution, these results underscore the usefulness of the diluted MHB for antimicrobial screening. Since bacterial mass will reduce with the dilution of the MHB media (Figure S3), it is important not to dilute the media to the extent where bacteria do not grow or differences in bacterial growth are no longer clear.

Application of the diluted MHB medium for antimicrobial peptide screen.

We hypothesized that antimicrobial agents could better display their activity in the diluted MHB medium, thereby increasing positive hits during antimicrobial screen. To illustrate this, we synthesized a small library of overlapping LL-37 peptides with 8–10 amino acids, covering the entire length of LL-37 (Figure 3). Select peptide parameters such as net charge and hydrophobic percentage are provided in Table 3 and additional parameters are listed in Table S1. These fragments are all shorter than KR-12 (12 residues), the smallest antibacterial peptide against E. coli previously identified in rich media (100%).¹⁵ In 100% MHB, all these shorter peptides, including LL-37 and KR-12, did not display any activity against S. aureus USA300 (Figure 3). In the case of E. coli, LL-37 and KR-12 were active. Most of the ultrashort peptides were inactive in rich media (MIC > 64 μM). RIK-10, however, emerged as a new peptide that inhibited the growth of E. coli in rich media (MIC 16 μM). RIK-10 is a 10-residue peptide corresponding to residues 23–32 of LL-37 in Figure 3. It is named RIK-10 here to distinguish it from the inactive RI-10 peptide (corresponding to residues 19–28 of LL-37) we previously reported. In 12.5% MHB, however, LL-37, KR-12, and RIK-10 all became active against S. aureus USA300. In the same diluted medium, KR-8 (an eight residue LL-37 peptide starting with KR) became active against E. coli (MIC 8 μM) but not S. aureus (MIC > 64 μM). In addition, we observed very weak activity for LL-10 and RK-9 against E. coli (MIC 32–64 μM) (Figure 3). To further validate our screening condition, we also compared antimicrobial activity of these LL-37 peptides in RPMI and DMEM media (Table S2). LL-10, KE-10, LR-10, and RK-9 were not active (MIC>64 μM). In DMEM, RIK-10 was active against E. coli (MIC 8 μM), although it showed poor activity in RPMI or against S. aureus. KR-8 only showed a poor activity against E. coli in DMEM. LL-37 and KR-12 remained active in both RPMI and DMEM against S. aureus and E. coli. Collectively, we obtained similar trends for these LL-37 peptides in diluted MHB or in cell culturing media. Such a picture is consistent with our previous finding that the C-terminus of LL-37 is disordered and not involved in bacterial killing, while the N-terminus of LL-37 is weakly active.¹⁶ The major antimicrobial region is located in the central region of LL-37.

Figure 3.

Antimicrobial screening of ultrashort LL-37 fragments in 12.5% MHB led to the identification of KR-8 and RIK-10 (peptide activity in μM). EC12.5% means anti-E. coli assay in 12.5% MHB, while SA12.5% stands for anti-S. aureus assay in 12.5% MHB. Likewise, 100% means rich MHB without dilution.

Table 3.

Enhance the Activity (μM) of KR-8 into LL-37mini with Eight Amino Acids¹

Peptide	Amino acid sequence	Q	Pho	S. aureus	E. coli	P. aeruginosa	K. pneumoniae	A. baumannii
W538	KRIVQRIK	+5	38%	>32	>32	>32	>32	>32
W539	KRIWQRIK	+5	38%	>32	>32	>32	>32	>32
W540	KRIWQRWK	+5	38%	>32	>32	>32	>32	>32
W541	KRWWQRWK	+5	38%	>32	8	>32	>32	>32
W542	KRWWQWWK	+4	50%	8	4	4–8	>32	32
W543	RRWWRWWR	+5	50%	4–8	8	8	>32	>32
W544	RRWWRWWL	+4	63%	4	4	4	8–16	8–16

¹All the peptides are C-terminally amidated. Q: net charge; Pho: hydrophobic content; KR-8: W538 in this series; W543: LL-37mini.

Interestingly, KR-8 and RIK-10 are both derived from the core antimicrobial region of human LL-37 we previously identified via NMR studies (FK-16, Figure 3).¹⁶ An N-terminally glycine-appended FK-16 called GF-17 is active against both E. coli and S. aureus in both rich and diluted MHB media (Figure 3). The addition of a glycine at the N-terminus has a minimal impact on the peptide since FK-16 and GF-17 have similar antimicrobial potency.¹⁷ Like the activity spectrum of KR-12 in rich media, KR-8 was also active against E. coli but not S. aureus in 12.5% MHB, identifying an even shorter anti-E. coli region within LL-37 (Figure 3). This KR-8 peptide (residues 18–25 of LL-37) identified in a more sensitive medium is four residues shorter than KR-12 (residues 18–29 of LL-37).

The ultrashort peptide KR-8 provides a template for engineering LL-37mini.

Since KR-8 (i.e., W538 in Table 3) is the shortest active template, we next aimed to increase peptide activity. A series of peptides (W538-W544) was designed (Table 3) by replacing aliphatic amino acids with fused aromatic residues. Antibacterial activity of these peptides is also listed in Table 3. After the deployment of three tryptophan (W) residues, we first observed activity for W541 against E. coli (MIC 8 μM). When one more W was used to substitute R23 (named as in LL-37, Figure 1), the resulting peptide W542 gained activity against S. aureus and P. aeruginosa. Due to a preferred association between W and R in Trp-rich peptides,¹⁸ we then generated an arginine-rich peptide by changing Q22 to R22 and two K to R. These changes had only a subtle effect on peptide activity. Also, the peptide became active against additional strains K. pneumoniae and A. baumannii when the C-terminal residue was replaced with a leucine residue. Since the net charges of these peptides are relatively constant, it is the increase in hydrophobic amino acids of the KR-8 template to 50% that was critical to enhance peptide antibacterial activity against S. aureus USA300, E. coli 423–17, and P. aeruginosa 423–17 (Table 3). In addition, the increase in peptide hydrophobic ratio from W541 (38%, only E. coli), W543 (50%, three bacteria), to W544 (63%, five bacteria) proportionally increased the activity spectrum of the peptides (Table 3).

We then evaluated hemolytic activity of this series of KR-8 derived peptides to human red blood cells (Figure 4A). Among the seven peptides, only two peptides (W540 and W544) displayed peptide-dose dependent hemolysis till 200 μM. W544 caused 50% hemolysis (HC₅₀) at about 80 μM, while it took twice the amount of W540 at ~ 150 μM to reach 50% hemolysis. Hence, W544 was more hemolytic than W540, consistent with a high hydrophobic content of W544 at 63% (Table 3). Different from W540, peptides W541-W543 were not hemolytic at least till 200 μM, implying isoleucine at position 3 played an important role in the hemolytic ability of W540 (Figure 4A). We also tested peptide toxicity to human HaCaT skin cells (Figure 4B). Similar to our finding in hemolytic assays (Figure 4A), W540 and W544 were more toxic than other peptides (Figure 4B). W543 was least toxic to HaCaT cells, similar to daptomycin. As a consequence, W543 was selected as a candidate for additional studies. Because W543 is engineered based on the shortest active peptide KR-8 of human LL-37, we referred to W543 hereinafter as LL-37mini, a miniature version of LL-37. LL-37mini is composed of four aromatic Trp amino acid, closely mimicking the four phenylalanine residues of human LL-37 that all bind bacterial anionic lipid.¹⁵

Figure 4.

Cytotoxicity of a series of peptides W538-W544 designed based on KR-8 (i.e., W538) by hydrophobic substitution: (A) Hemolysis of human red blood cells and (B) Viability of human skin HaCaT cells due to the treatment of W538-W544 at various peptide concentrations. Daptomycin was included as a control. The results were expressed as mean ± standard deviation.

We then evaluated the effects of salts, pH and human serum on the antibacterial activity of LL-37mini against MRSA in MHB. A decrease of pH from 7.2 to 6.3 increased MIC by twofold. The addition of 150 mM NaCl did not alter the MIC. It appeared that both acidic pH and physiological salts had a minimal impact on peptide activity (Table 4). However, 10% human serum reduced the activity of LL-37mini by 8-fold. Likewise, serum also influenced the activity of 17BIPHE2. Among the antibiotics tested, only the MIC values of amikacin and muporicin increased four-fold in 10% human serum (Table 4). Previously, we found that multiple human serum proteins could associate with C10-KR8 where KR-8 is conjugated with a C10 fatty acid at the N-terminus.¹⁹ Since serum binding is an inherent property of human LL-37,²⁰ all the above peptides retained this property.

Table 4.

Comparison of Antimicrobial Robustness of LL-37mini with Antibiotics in MHB¹

Antibiotic	pH 7.2	pH 6.3	NaCl (150 mM)	10% Serum	20% Serum
LL-37mini	8	16	8	64	ND
17BIPHE2	4	16	4	>64	ND
Linezolid	8	8	8	8	8–16
Amikacin	8	32	32	32	32–64
Gentamicin	0.5	1–2	1	0.5–1	0.5
Tobramycin	2	4	4	2–4	2
Muporicin	1	0.5	1	4	4
Daptomycin	1	1	1	1	1
Nafcillin	4–8	16	32	16	16–32
Vancomycin	0.5	0.5	0.5–1	0.5	0.5–1

¹Antimicrobial activity values are in μM; ND, not determined.

We also measured bacterial killing kinetics of LL-37mini against S. aureus USA300. While bacterial CFU increased in the untreated culture, it decreased with time and was not detected at 120 min due to LL-37mini treatment at 2× MIC (Figure 5A). Likewise, S. aureus was also killed in 2 h by daptomycin despite at a slower rate compared to LL-37mini. LL-37 (Figure 1) is known to target bacterial membranes.^21,22 To verify this, we conducted membrane permeabilization experiments for LL-37mini in the presence of propidium iodide (PI). In this experiment, one does not anticipate fluorescence increase when S. aureus USA300 was not treated, indicating an intact membrane (Figure 5B). As a negative control, no fluorescence increase was observed for gentamicin treated MRSA, either, since this antibiotic inhibits protein synthesis. However, there was a clear fluorescence increase when treated with LL-37mini, indicative of membrane damage. As a positive control, daptomycin only showed membrane permeabilization after a delay as observed previously.²³ We also investigated the membrane permeation of E. coli by LL-37mini. Like the case of MRSA, gentamicin did not permeate the E. coli membrane. However, colistin was a powerful membrane permeator. In the case of LL-37mini, there appeared to be a 30 min delay in membrane permeabilization (Figure S4A). Cationic AMPs may also depolarize bacterial membranes.¹⁹ Triton X-100 as a positive control showed strong membrane depolarization of S. aureus USA300. However, LL-37mini did not show a clear effect at the highest concentration (2× MIC) we tested (Figure 5C). In the case of E. coli, LL-37mini showed some depolarization (Figure S4B). We then observed the cells of S. aureus USA300 with and without peptide treatment using SEM. While the cells were smooth and spherical without treatment (Figure 5D), cell damages were evident after treatment with LL-37mini at 2× MIC (Figure 5E).

Figure 5.

Killing kinetics and mechanism of action of LL-37mini and bacterial resistance development. (A) LL-37mini (2× MIC, 16 μM) killed the exponential phase of S. aureus USA300 LAC in 120 min, similar to daptomycin. (B) Permeabilization and (C) depolarization of the membranes of S. aureus USA300 LAC by LL-37mini treated at 1 × MIC of W543 (8 μM), daptomycin (1 μM), and gentamicin (1 μM). RFU: Relative fluorescence intensity. The scanning electron microscopy (SEM) images of untreated (D) and LL-37mini-treated (E) S. aureus. (F) Resistance development of S. aureus USA300 LAC in a multiple passage experiment in the presence of sub-MIC levels of LL-37mini (green rectangle), nafcillin (blue diamond), and daptomycin (golden triangle). Results were expressed as mean ± standard deviation. Resistance development is indicated by a substantial increase in MIC.

It is proposed that membrane damage makes it difficult for bacteria to develop resistance.¹⁹ To verify this, we conducted a multiple passage experiment (Figure 5F). After 17 days, there was essentially no change in the MIC values of LL-37mini. In the same experiment, MRSA did not develop resistance to daptomycin, either. However, the MIC of nafcillin increased to 128 μM (32 fold), indicating rapid resistance development.

Antibiofilm activity of LL-37mini in vitro and in vivo.

Since biofilms are resistant to antibiotics, we also tested the capability of LL-37mini in inhibiting bacterial attachment, biofilm formation, and disrupting the preformed biofilms. Surface attachment is the initial step for biofilm formation. In a peptide dose-dependent assay, LL-37mini did not show anti-attachment ability at 4× and 8× MIC. However, daptomycin did (Figure 6A). However, LL-37mini was more effective than daptomycin in inhibiting biofilm formation (Figure 6B). Moreover, a dose-dependent effect was observed against the 24 h preformed biofilms of S. aureus USA300 (Figure 6C). At 32 μM, the MRSA biofilms were essentially eliminated by LL-37mini. Better disruptive effects than daptomycin were also observed for LL-37mini against 48 or 72 h preformed biofilms of S. aureus (Figure 6, D & E). We also tested the antibiofilm activity of LL-37mini against E. coli E423–17 treated at varying doses. The peptide appeared to be very effective against 24-h preformed biofilms as it reduced live bacterium by ~40% when treated at 8 μM (Figure S5). At 16 μM, live E. coli cells were almost eliminated. These experiments proved the antibiofilm efficacy of LL-37mini in vitro.

Figure 6.

Antibiofilm activity of LL-37mini against S. aureus USA300 in vitro and in vivo. (A) Bacterial attachment (B) biofilm inhibition, (C-E) disruption of biofilms formed for 24, 48 and 72 h, respectively, in 100% TSB media and (F) antibiofilm efficacy of LL-37 in murine wounds. S. aureus USA300 biofilms were formed in the wounds created on the back of BALB/c mice for 24 h followed by treatment with water (untreated control), LL-37 mini (4.4 mg/kg per wound), or daptomycin (10 mg/kg per wound) for 24 h. Results were expressed as mean ± standard deviation.

Since LL-37mini is potent and not toxic, we then tested its antibiofilm efficacy in vivo using a murine wound model. This study was approved by the University of Nebraska Medical Center (UNMC, see Method). Wounds were created and infected with S. aureus USA300 at a CFU of ~1×10⁸ and biofilms were allowed to form for 24 h. Without treatment, the MRSA count reached a CFU of below 10¹². This time suffices for biofilm formation.²⁴ In vitro, S. aureus can form biofilms in a couple of hours.²⁵ When the biofilms were treated with daptomycin (10 mg/kg per wound), the bacterial burden was reduced by ~3 logs. When treated with LL-37mini at 4.4 mg/kg per wound, we observed a bacterial CFU decrease by 2 logs (Figure 6F). Hence, LL-37mini could eliminate MRSA in wound biofilms similar to daptomycin (positive control).

Finally, we also compared antibacterial activity and stability of the above LL-37mini form (L-form) with that made of D-amino acids. The D-form showed the same MIC as the L-form (made of L-amino acids), consistent with membrane targeting.²³ However, the L-form started to lose activity in the presence of proteases such as trypsin and chymotrypsin. In contrast, the D-form remained active even after incubation with these two proteases (Figure 7). Interestingly, both L and D-forms of LL-37mini retained antibacterial activity against S. aureus USA300 after incubation with pepsin (Figure 7). It seems that LL-37mini (L-form) is more stable than GF-17 (L-form), which was degraded by pepsin under the same condition.²⁵

Figure 7.

Peptide stability evaluation with and without proteases. In this assay, growth curves of S. aureus USA300 LAC were plotted with and without protease pre-treatment of L and D-forms of LL-37mini: (A) without protease (no growth at 8 μM), (B) trypsin, (C) chymotrypsin, and (D) pepsin at a peptide:protease molar ratio of 40:1. Peptides were incubated with proteases at 37°C for 1 h before mixing with the bacterial culture. Results were expressed as mean ± standard deviation. This experiment is good for initial stability screening of peptides.¹²

DISCUSSION

LL-37 is an important human host defense peptide known to inhibit various pathogens, ranging from viruses and fungi to bacteria.¹⁷ Selection of a proper medium is important during antimicrobial assays. Our study revealed that the standard MHB medium can be used for antimicrobial screening against E. coli and A. baumannii. This is because the MIC values for E. coli and A. baumannii are least influenced by media dilution (Tables 1 & 2). In rich media, KR-12 was previously identified as the smallest antibacterial peptide of human cathelicidin LL-37 against E. coli but not S. aureus USA300.¹⁵ Using overlapping synthetic peptides with 12 residues, Jenssen and colleagues also found KR-12 to be active.²⁶ This study screened a library of even smaller peptides (8–10 residues) covering the entire length of LL-37 and uncovered the activity of RIK-10 against E. coli in rich media. To our knowledge, RIK-10 is the newly identified smallest active peptide of human LL-37 discovered to date in rich media.

However, we and others^11,13 frequently failed to observe antimicrobial activity of LL-37 against S. aureus due to the interaction of LL-37 with anionic components in rich media. In this study, we identified a diluted MHB for detecting the activity of LL-37 against S. aureus (Table 1). Because this diluted medium is only utilized at the last treatment stage, bacterial culturing and biofilm growth in 100% MHB are as efficient as usual. In 12.5% MHB, LL-37 was found as effective against MRSA as our engineered peptide 17BIPHE2. Also, KR-12 was as active as its parent peptide LL-37 (Figure 3). In addition, the majority of antibiotics retain the same MIC with the dilution of MHB (Figure S2). In diluted MHB, an even shorter peptide KR-8 (8 amino acids) is identified as the minimal antibacterial peptide against E. coli (Figure 2). This medium is also useful for screening activity against S. epidermidis, P. aeruginosa, and K. pneumoniae owing to increased antimicrobial susceptibility (Table 2). We obtained similar screening results for LL-37 ultrashort peptides in DMEM and RPMI media (Table S2). It is conceivable that the screening sensitivity can be further increased if the antimicrobial assay is conducted using a sensitive bacterial strain such as S. aureus mprF knockout strain.²⁷ The loss of a functional mprF gene disabled the lysine coating on the membrane surface of MRSA,²⁸ making the strain more susceptible to killing by cationic peptides such as LL-37 (Figure S6). Like KR-12 and RIK-10 in rich media, KR-8 only retained activity against Gram-negative E. coli in diluted MHB. It is interesting to note that both KR-8 and RIK-10 are the fragments of the core antimicrobial region (FK-16) of LL-37 (Figure 3). Both ultrashort peptides remain active against E. coli in the DMEM medium used to culture mammalian cells (Table S2). Remarkably, FK-16 is active against both E. coli and S. aureus in rich and diluted MHB. These new results justified our initial selection of the glycine capped FK-16 template (i.e., GF-17) to engineer 17BIPHE2 into a potent, stable, and selective peptide to eliminate the ESKAPE pathogens.¹² Taken together, this study shines new light on sequence-activity relationship of human LL-37 since the two even shorter active peptides KR-8 and RIK-10 discovered herein in a more sensitive medium correspond to the core antimicrobial region we discovered previously.¹⁶

There has been a strong desire to develop human cathelicidin into a new antibiotic. Both library and structure-based design have been utilized (for a review, see ref¹⁷). It is important to search the smallest antimicrobial regions from LL-37 to bring down the production cost. Based on the 3D structure of LL-37 (Figure 1), one would obtain LL-31 if the disordered C-terminus is omitted. Indeed, LL-31 showed potent activity against multiple bacterial pathogens.^29,30 LL-31, corresponding to the long helix in the 3D structure of LL-37 (Figure 1),¹⁵ consists of two hydrophobic domains split by serine 9. When the N-terminal hydrophobic domain is removed, we recently obtained SK-24 (Figure 1).³¹ Interestingly, this peptide was found to be most helical in phosphate buffer. However, it is the least toxic antibacterial peptide compared to other LL-37 peptides investigated therein. Based on NMR studies, we also identified the major anticancer and antibacterial region, FK-16/GF-17, initially called LL-37(17–32) (Figure 1).¹⁶ 17BIPHE2, engineered based on GF-17, is stable, selective and potent against the ESKAPE pathogens,^12,24 Ebola viruses,³² and sperms to avoid unwanted pregnancy.³³ KR-12, the smallest antibacterial peptide of LL-37 identified in rich media (Figure 1),¹⁵ has been used as a template for designing various antimicrobial candidates, ranging from linear to cyclic peptides.^34,35 KR-12 has also been lipidated to become lipopeptides.^19,36 It was notable that C10-KR8, consisting of KR-8 in conjugation with a capric acid at the N-terminus, was identified as the optimal antimicrobial via screening a lipopeptide library generated from systematically altering both the peptide and fatty acid chain lengths.¹⁹ This study has advanced the design of LL-37 by identifying ultrashort active peptide templates such as KR-8 (residues 18–25 of LL-37, Figure 1) under a more sensitive screening condition. Considering the cost advantage of ultrashort peptides, we also succeeded in engineering KR-8 into LL-37mini, a potent, selective LL-37 derived antimicrobial peptide with the shortest length and without conjugation with other molecular moieties. LL-37mini also gains stability to proteases when synthesized using D-amino acids (Figure 7). Our design of LL-37mini was guided by our structural knowledge. The deployment of four fused aromatic residues was inspired by our observation that all the four phenylalanine residues of LL-37 directly interdigitate into bacterial membranes to interact with anionic phosphatidylglycerol.¹⁵ LL-37mini has the desired activity spectrum against both S. aureus and P. aeruginosa (Table 3), two major pathogens in chronic wounds. This study laid the foundation for investigating the antimicrobial and wound healing properties of LL-37mini incorporated into nanofibers. When formulated, even the L-form of LL-37mini will work well.²⁵ Collectively, this study has expanded our knowledge on the structure-activity relationships of human LL-37 and enriched our reservoir of LL37-derived antimicrobials to combat drug-resistant pathogens.

CONCLUSION

A caveat in antimicrobial screening is failure to detect active candidates. This study has identified a diluted MHB medium that enabled the observation of antibacterial and antibiofilm activity of human cathelicidin LL-37 against S. aureus USA300. By screening a small library of ultrashort peptides in 12.5% MHB, we were able to identify two active peptides (KR-8 and RIK-10) within LL-37. Notably, KR-8 and RIK-10 correspond to the core antimicrobial region of LL-37 we initially discovered from NMR structural studies.¹⁶ Shorter peptides are desired for antimicrobial development due to reduced production cost. We then demonstrated that the antimicrobial activity of the KR-8 template could be enhanced via peptide engineering. The engineered peptide LL-37mini is both potent and selective. It also showed antibiofilm efficacy in vitro and in vivo. LL-37mini constitutes a novel lead for developing new antimicrobial and antibiofilm agents. We propose that the diluted medium obtained here is useful for initial antimicrobial screen to identify antimicrobials from natural sources or artificial libraries. Antibacterial assays in such a medium will increase positive hits against numerous bacteria, including S. aureus, S. epidermidis, P. aeruginosa, and K. pneumoniae.

METHODS

Chemicals and Peptides.

All the chemicals were purchased from established vendors such as Fisher and Sigma. Peptides were made by Genemed Synthesis, Inc. (San Antonio, TX). All the peptides were highly purified and reached over 95% purity based on HPLC. The correct mass of each peptide was validated by Mass Spectrometry. Peptides were quantitated by measuring UV absorbances at 280 nm (with W) or at 215 and 225 nm (without W) based on the Waddell’s method.³⁷

Antibacterial Assay.

The following resistant bacterial clinical strains were used in this study: S. aureus USA300 LAC, S. epidermidis 1457, E. coli E423–17, P. aeruginosa E411–17, K. pneumoniae E406–17, and A. baumannii B28–16. While the S. aureus USA300 strain is methicillin resistant, most of the Gram-negative bacteria contain extended-spectrum beta-lactamase (ESBL) and are resistant to beta-lactam antibiotics. Peptide activity against bacteria were tested using the established lab protocol as described previously.¹⁸ In brief, a peptide concentration gradient with twofold dilution was made in the 96-well polystyrene microplate at 10 μL per well. Bacteria were grown to the exponential phase (i.e., optical density at 600 nm ≈ 0.5), diluted to ~10⁵ CFU/mL in different percentages of MHB, TSB, or LB, and partitioned into the 96-well microplate at 90 μL per well. Rich media prepared as suggested by the vendors are defined as 100% in this study. They were diluted with autoclaved distilled water to obtain different percentages. The microplates were incubated at 37°C overnight and read on a ChroMate 4300 Microplate Reader at 600 nm (GMI, Ramsey, MN). The peptide concentration in the wells without bacterial growth is the minimal inhibitory concentration (MIC). As the convention, MIC values were represented as ranges.

For pH effects on peptide activity, the pH of the medium was adjusted to a targeted pH for autoclave and then remeasured at room temperature. For salt and serum effects on peptide activity, stock NaCl solution or human serum were added to the media.

Peptide Protease stability.

Protease stability of the peptides was tested in the presence and absence of mammalian trypsin, chymotrypsin, and pepsin (Thermo Fisher Scientific, MA) based on a modified antimicrobial assays, which give a similar conclusion as HPLC-based stability assays.²⁵ A solution of peptide/protease (40:1 molar ratio) was made in phosphate buffer saline (PBS; pH 7.1, Gibco, NY) and incubated at 37°C for 1 h. As controls, peptides without the proteases were also incubated in the same manner. Then, aliquots of 10 μL were transferred to a 96-well polystyrene microplate and mixed with the exponential phase MRSA USA300 culture as performed in antibacterial activity assays above. If the peptide is stable, no growth is anticipated at and above the MIC.

Killing kinetics.

MRSA USA300 was grown to the exponential phase (OD₆₀₀ ~ 0.3) in MHB. The culture was diluted to OD₆₀₀ 0.001 (~10⁵ CFU/mL). One milliliter of bacterial suspension was then mixed with LL-37mini or daptomycin at 2× MIC and incubated at 37°C. After 15, 30, 60, 90, and 120 min incubation, 50 μL was taken and serially diluted with 1×PBS. Then, 50 μL of the diluted suspension was plated on Mannitol Salt Agar plates (NEOGEN, MI). The plates were incubated overnight at 37°C for bacterial CFU determination the next day.

Hemolytic Assay.

The hemolytic effects of peptides were evaluated using a previous method with minor modifications.¹⁹ Human red blood cells (hRBCs, UNMC Blood Bank) were washed (3×) with blood bank saline (isotonic solution 0.90% w/v, Fisher). Cells were diluted to 2% using the saline solution and aliquots of 90 μL were added to 96-well polystyrene microplate containing 10 μL of serially diluted peptide solutions. After incubation at 37°C for 1 h, plates were centrifuged at 500g for 5 minutes. Aliquots of the supernatant were transferred to a new 96-well microplate. The amount of hemoglobin released was measured at 545 nm using a ChroMate Microplate Reader. To calculate the percent lysis, it was assumed that 100% of the hemoglobin was released when hRBCs were treated with 1% Triton X-100, while incubation with water resulted in 0% release. HC₅₀ is defined as the concentration of the peptide that caused 50% lysis.

Cell Viability Assay.

Human keratinocytes HaCaT cells were grown in Dulbecco’s Modification of Eagles’s Medium/High Glucose (DMEM, Hyclone, UT) containing 10 % fetal bovine serum (FBS) (Mediatech, Corning, Manassas, VA). Cells were grown at 37°C in 5% CO₂, and the medium was changed every other day. Peptides were diluted in a 96-well polystyrene microplate (10 μL each well) and mixed with 90 μL of cell suspensions (10⁵ cells/mL) in their respective media. The mixture was incubated at 37°C in a 5% CO₂ for 24 h. Then, 20 μL of MTS reagent (MTS cell proliferation assay kit, Promega, WI) was added to each well and incubated at 37°C for 2 h. The absorbance was determined at 492 nm using a microplate reader. The percentage viability was determined using the following formula: Percentage viability = (OD value of treated cells/OD value of untreated cells) × 100, where OD is the optical density.

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 TSB 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.

Membrane Depolarization.

An overnight culture of MRSA USA300 was inoculated into TSB and grown to exponential phase. Bacteria were washed with 1×PBS, re-suspended in twice the volume of 1×PBS containing 25 mM glucose, and incubated at 37°C for 15 minutes. Then, 500 nM (final concentration) of the DiBAC4 (3) bis-(1, 3-dibutylbarbituric acid) trimethine oxonol (ANASPEC, CA) was added and vortexed gently. Aliquots of 90 μL of the energized bacteria solution were loaded to the 96 well plates (Corning COSTAR, AZ) and placed in a FLUOstar Omega microplate reader (BMG LABTECH, NC). Fluorescence was read for 20 min at excitation and emission wavelengths of 485 nm and 520 nm, respectively. Then, 10 μL of peptide solutions was added and fluorescence readings were recorded for 40 min. Triton X-100 (0.1%) was used as a positive control.

Scanning electron microscopy (SEM).

The exponential phase S. aureus USA300 LAC was treated with 2× MIC of LL-37mini for 30 min. Samples were washed three times with 1× PBS, and fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M PBS. Next, the samples were washed with Sorensen’s Buffer and post-fixed in 1% osmium tetroxide in water for 30 min. All samples were then washed in Sorensen’s buffer and dehydrated through a graded ethanol series (50%, 70%, 90%, 95%, 100% with × 3 changes). Subsequently, samples were placed in HMDS 100% for 10 min with 3 changes and left in HMDS in open dishes in the fume hood overnight to allow the HMDS to evaporate. The following day, the samples were mounted on 25 mm aluminum SEM stubs with carbon adhesive tabs. Silver paste was placed around the edges of the samples. Samples were Sputter Coated with 50 nm of gold/palladium in a Hummer VI Sputter Coater (Anatech Ltd., Battle Creek, MI) and examined in a FEI Quanta 200 SEM at the UNMC Electron Microscopy Core Facility.

Bacterial resistance development.

The experiment was conducted as described previously.³⁸ Briefly, MRSA USA300 was grown to the exponential phase in MHB and diluted to OD₆₀₀ 0.001 (~10⁵ CFU/ mL). Aliquots of 90 μL were added to 96-well polystyrene microplate (Costar, Corning, NY, USA) containing 10 μL of serially diluted LL-37mini, nafcillin, or daptomycin. After overnight incubation at 37°C, the plates were read at 600 nm using a ChroMate Microplate Reader (GMI, Ramsey, MN, USA). Untreated bacterial culture and medium were included as untreated control and blank, respectively. The minimal inhibitory concentration (MIC) is the lowest peptide concentration that inhibited the bacterial growth. The wells with sub-MIC levels of the peptides that retained growth approximately half the growth of the control wells were re-inoculated in fresh MHB with sub-MIC concertation of peptides or antibiotics to attain exponential phase for MIC determination. In total, 17 passages of the bacteria cultures were conducted. A rapid increase in MIC is an indication of antibiotic resistance.

In vitro antibiofilm activity of LL-37mini.

Inhibition of bacterial attachment.

Bacterial attachment is the initial step for biofilm formation. For this experiment, an overnight culture of MRSA USA300 was grown overnight in TSB media to an optical density (OD₆₀₀) of ~1.0. Then, 180 μL of this culture were added to each well of the microtiter plates containing 20 μL of various MIC folds (1×, 2×, 4×, and 8× MIC) of LL-37mini or daptomycin solutions. The plates were then incubated at 37°C for 1 h. Next, media was aspirated, washed with 1× PBS, and 200 μL of TSB containing XTT 10% [2, 3-bis (2-methyloxy-4-nitro-5-sulfophenyl)-2H-tertazolium-5-carboxanilide] (ATCC, VA, USA) solution was added. After incubation at 37°C for 2 h, absorbance was read at 450 nm using a ChromateTM microtiter plate reader. TSB containing 10% XTT served as blank, while bacterial culture treated with water served as a positive control. Percentages of viable cells in biofilms were plotted by assuming 100% growth in water treated control.

Biofilm inhibition.

MRSA USA300 (10⁵ CFU/mL) was prepared from the exponential phase. Aliquots of 180 μL of bacterial suspension in TSB were added to each well of the microtiter plates containing 20 μL of various MIC folds (0.5 to 2× MIC) of LL-37mini or daptomycin solutions and incubated at 37°C for 24 h. Media were then pipetted out and the wells were washed with 1× PBS to remove planktonic cells. Live cells in biofilms were quantitated by XTT as described above.

Effects of Peptides on Established Biofilms in Vitro.

Bacteria (S. aureus USA300 LAC and E. coli E423–17) were grown up in rich TSB media overnight. A second inoculation was made the next day to reach an exponential phase (OD₆₀₀ ~0.4). Microtiter plates (96 wells, Corning Costar Cat No. 3595), after aliquoting with 180 μL of the culture to each well, were incubated at 37°C for 24 h to form biofilms in rich TSB. Media were aspirated post incubation and the attached biofilms were washed with 1× PBS to remove the planktonic bacteria. Each well was aliquoted with 20 μL of 10× peptide solution and 180 μL of 10% fresh MHB, and plates were further incubated at 37°C for 24 h. Biofilms treated with water served as the positive control while media without bacterial inoculation served as the negative control. Live cells in the biofilms were quantitated using the XTT as described above. Absorbance was read at 450 nm (only media with XTT containing wells served as the blank) using a ChromateTM microtiter plate reader. Percentage biofilm growth for the peptide was plotted by assuming 100% biofilm growth in bacterial control alone. The data were represented as mean ± SD, plots were generated using GraphPad prism 7, where * indicates p<0.05, ** p<0.01, and ***P<0.001, and ****p<0.0001 (one-way analyses of variance).

In Vivo Anti Biofilm Activity of LL-37mini.

Preparation of Inoculum.

An overnight culture of S. aureus USA300 LAC was inoculated in fresh MHB and incubated at 37°C for 3 h. The suspension was centrifuged at 3,000 g for 5 min and the supernatant was discarded. The bacteria were re-suspended in 1×PBS and the OD₆₀₀ was adjusted to 0.6 (~3 × 10⁷ CFU/mL).

Experimental Animals.

We have established an alternative murine wound model slightly different from our previous studies.^24,39 Female BALB/c mice (3−4 weeks, ~20 g) were fed with standardized food (Teklad Laboratory diet for rodents) and water (Hydropac^® Alternative Watering System) ad libitum. Mice were kept in ventilated cages (IVCs) at a temperature of 20–24°C, humidity of 50–60%, 60 air exchanges per hour, and a 12/12-hour light/dark cycle. All materials, including IVCs, lids, feeders, bottles, bedding, and water, were autoclaved before use. All animal manipulations were performed in a class II laminar flow biological safety cabinet. The study was approved by the Institutional Animal Care and Use Committee (IACUC) of UNMC (Protocol no. 22-015-08-FC).

In Vivo Biofilm Assay.

Mice were anesthetized by intraperitoneal injection of ketamine-xylazine (100 mg/kg + 10 mg/kg). The dorsal back hair was removed with a clipper and depilatory cream, followed by cleaning the shaved area with isopropyl alcohol (70%) and povidone iodine swabs. Two full-thickness skin wounds with a diameter of 6 mm were then created using a disposable biopsy punch (Integra Miltex, MA). The wounds were immediately inoculated with MRSA USA300 (10 μL of ~3 × 10⁷ CFU/mL) and wounds were covered with a transparent film dressing (3M Tegaderm^™, Deutschland GmbH, Neuss, Germany). Buprenorphine (0.5 mg/kg, SC) was injected and mice were kept in individual cages. After 24 h of inoculation and biofilm formation, treatment groups received 3×10 μL LL-37mini or daptomycin dissolved in sterile water. Treatment was applied topically on the wound and wounds were covered with the transparent film dressing. The infected control group received an equal volume of sterile water. After 24 h from the time of treatment, mice were euthanized under CO₂ and specimens were collected using sterile 8 mm biopsy punch (Integra Miltex, MA, USA) into sterilized tubes containing 2 mL of PBS. Tissues were blended using a homogenizer, diluted further in 1×PBS and plated on mannitol salt agar plates. Finally, the plates were incubated at 37°C for 20 h and the CFUs were counted.

Statistical Analysis.

Data were analyzed using GraphPad Prism 7 (GraphPad Software, Inc., San Diego, CA, USA) and values were expressed as mean ± standard deviation. One-way analyses of variance (ANOVA) were used to compare the mean values among treatment groups. P < 0.05 was considered statistically significant.

ABBREVIATIONS

CFU

colony forming units

DMEM

Dulbecco’s Modified Eagle Medium

KR-8

minimal antibacterial peptide of LL-37 newly discovered in diluted MHB

KR-12

minimal antibacterial peptide of LL-37 identified in rich media

LL-37

a 37-residue human innate immune peptide starting with a pair of leucine amino acid

LL-37mini

the minimal antibacterial peptide designed based on the KR-8 fragment of human LL-37

LB

Luria broth

MHB

Mueller Hinton Broth

MIC

minimum inhibitory concentration

MRSA

methicillin-resistant Staphylococcus aureus

RPMI

Roswell Park Memorial Institute

TSB

tryptic soy broth

SEM

scanning electron microscopy.