Resurrecting Inactive Antimicrobial Peptides from the Lipopolysaccharide Trap

Abstract

Host defense antimicrobial peptides (AMPs) are a promising source of antibiotics for the treatment of multiple-drug-resistant pathogens. Lipopolysaccharide (LPS), the major component of the outer leaflet of the outer membrane of Gram-negative bacteria, functions as a permeability barrier against a variety of molecules, including AMPs. Further, LPS or endotoxin is the causative agent of sepsis killing 100,000 people per year in the United States alone. LPS can restrict the activity of AMPs inducing aggregations at the outer membrane, as observed for frog AMPs, temporins, and also in model AMPs. Aggregated AMPs, “trapped” by the outer membrane, are unable to traverse the cell wall, causing their inactivation. In this work, we show that these inactive AMPs can overcome LPS-induced aggregations while conjugated with a short LPS binding β-boomerang peptide motif and become highly bactericidal. The generated hybrid peptides exhibit activity against Gram-negative and Gram-positive bacteria in high-salt conditions and detoxify endotoxin. Structural and biophysical studies establish the mechanism of action of these peptides in LPS outer membrane. Most importantly, this study provides a new concept for the development of a potent broad-spectrum antibiotic with efficient outer membrane disruption as the mode of action.

INTRODUCTION

The cell wall of bacteria is essential for their survival. The cell wall of Gram-positive bacteria contains a thick layer of a polymer, termed peptidoglycan. The coarse meshwork of peptidoglycan provides a structural support, as well as balances osmotic pressure of the cytosol and the environment. In contrast, cell walls of Gram-negative bacteria are typified by an asymmetric outer membrane and a thin peptidoglycan layer adjacent to the plasma or inner membrane. The inner leaflet of the outer membrane contains phospholipids similar to the cytoplasmic membrane, whereas the outer leaflet of the outer membrane is predominantly composed of lipopolysaccharide (LPS) (1, 2). The cell wall or peptidoglycan of Gram-positive bacteria does not maintain a permeability barrier due to its little resistance toward the diffusion of antibiotics and antibacterial agents (3–5). While LPS faces the external environment, it acts as a permeability barrier for the outer membrane of Gram-negative bacteria (3–5). Although molecules of ≤600 Da can freely diffuse through outer membrane; however, higher-molecular-mass compounds have limited access through the outer membrane (3–5). LPS has an amphiphilic structure that can be divided into three regions: a conserved lipid A part, a highly variable polysaccharide or O antigen, and a core oligosaccharide (1, 2). Lipid A domain, a hexa-acylated glucosamine-based phospholipid, anchors with the acyl chains of phospholipids of the inner leaflet of outer membrane. The core oligosaccharide domain is centrally located and is covalently bonded with lipid A and O antigen. Apart from its role as a permeability barrier, LPS is also known as an endotoxin, a potent inducer of innate immune system in humans (6–8). While in the bloodstream, LPS recognizes Toll-like receptor 4 in macrophages, inducing the production of variety of inflammatory agents, such as tumor necrosis factor alpha and interleukin-1β (9, 10). These inflammatory agents are necessary to clear bacterial infection. However, a dysregulated immune system, often caused by a severe Gram-negative infection, may result in the overproduction of inflammatory molecules, leading to lethal septic shock syndromes (9, 10). Septic shock accounts for nearly 100,000 deaths annually in developed nations, including the United States (11, 12). At present, treatment for septic shock is rather limited. Consequently, there is intense interest in developing drugs against sepsis (10, 13–15).

Host defense antimicrobial peptides (AMPs), vital components of the innate immune system, act as a first line of defense against invading pathogens in most organisms (16–18). AMPs are considered to be highly important as leads or potential therapeutics against drug-resistant bacterial pathogens (19–23). Bacterial cell lysis caused by AMPs is an outcome of disruption of the cytosolic membrane integrity. Cationic and hydrophobic characteristics of a vast majority of AMPs are suitable for the insertion and perturbation of anionic bacterial membranes (24–26). However, intracellular targets for AMPs have also been identified (27–29). Studies with model membranes, resembling the cytosolic membrane of bacteria, suggest that the AMP's mechanism of cell membrane perturbation may include barrel stave, torodial pore, carpet mode, or general interfacial mode of action (30–32). It has also been realized that the cell wall components of bacteria play important roles in insertion and translocation of AMPs toward cytosolic membrane (33–35). Several studies have demonstrated higher MICs of AMPs for Gram-negative bacteria compared to Gram-positive organisms (36–40). In particular, LPS of the outer membrane of Gram-negative bacteria has been found to be actively involved in the mode of action of AMPs (5, 41–46). Due to the LPS barrier, there are fewer antibiotic precursors against multiple-drug-resistant Gram-negative strains in the drug discovery pipelines (47, 48). One of the mechanisms the LPS-outer membrane uses to reduce the efficacy of AMPs is by inducing self-association or aggregations of peptides (49–52). LPS-induced aggregation of AMPs may result in their complete inactivation, as observed in temporins, a group of AMPs obtained from the skin of the European red frog and in designed peptides (50–52). Temporins are among the shortest AMPs demonstrating potential in antibacterial therapeutics (52, 53). However, a number of temporins, including temporin-1Ta (TA) and temporin-1Tb (TB), are only active against Gram-positive bacteria (50–52). A similar observation has been made in designed Lys/Leu-based AMP or KL12, whereby the peptide showed limited activity against Gram-negative bacteria (49). AMPs trapped in LPS in oligomeric states are unable to traverse or translocate through the outer membrane, possibly causing their inactivation (52, 54, 55). In the present study, we demonstrate that a short LPS binding peptide motif or β-boomerang motif, GWKRKRFG, designed in our laboratory (56, 57), rescues these inactive AMPs, including TA, TB, and KL12 peptide from the LPS outer membrane. To accomplish this, synthetic peptides containing a β-boomerang motif at the C terminus were constructed and then investigated for bactericidal activity, cell lysis, and mode of action. In light of drug resistance and LPS permeability concerns, the present study suggests that a β-boomerang LPS binding motif could be useful for the development of potent, salt-resistant, nontoxic, and broad-spectrum antimicrobial agents.

MATERIALS AND METHODS

Peptides and rhodamine-labeled peptides were synthesized commercially by GL Biochem (Shanghai, China) and further purified by a reversed-phase high-pressure liquid chromatography (Waters) using a C₁₈ column (300-Å pore size, 5-μm particle size). A linear gradient of acetonitrile-water mixture was used for the purification, and the peak eluting with highest purity was collected and freeze-dried. Escherichia coli O111:B4 LPS, E. coli O55:B5 fluorescein isothiocyanate (FITC)-LPS, acrylamide, and NPN (1-N-phenylnaphthylamine) were purchased from Sigma. The bacterial strains were obtained from the American Type Culture Collection (ATCC), Manassas, VA. SYTOX green was obtained from Invitrogen.

Antibacterial assay.

The MIC of hybrid peptides was determined by using a broth dilution method against four Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 13883, and Salmonella enterica ATCC 14028) and four Gram-positive strains (Bacillus subtilis, Staphylococcus aureus ATCC 25923, Streptococcus pyogenes ATCC 19615, and Enterococcus faecalis ATCC 29212). Mid-log-phase cultures of these bacterial strains were obtained either in Mueller-Hinton (MH) or Luria-Bertani (LB) broth and diluted to an optical density at 600 nm (OD₆₀₀) of 0.01 (∼10⁶ CFU/ml). Twofold serial dilutions of the peptides were carried out in 96-well polypropylene microtiter plates in a total volume of 50 μl at concentrations ranging from 200 to 0.1 μM. To this, 50 μl of the diluted cells in MH or LB broth was added, followed by incubation for 18 h at 37°C. Water in the place of peptides acted as a negative control. The MICs were computed spectrophotometrically, and the concentration at which there was complete inhibition of bacterial growth was considered the MIC of the peptide. In order to dissect bacterial killing and bacterial-growth-inhibiting activity, the wells before and after MICs were streaked onto MH agar plates, followed by incubation overnight at 37°C. Wells that did not show visible bacterial growth were compared to wells that were considered for MICs.

Hemolytic assay.

Blood was collected from healthy mice in a tube containing EDTA. Red blood cells (RBCs) with EDTA were centrifuged at 800 × g for 10 min to remove the buffy plasma coat layer. Then, the resulting RBCs were resuspended in phosphate-buffered saline (PBS; 35 mM phosphate buffer, 150 mM NaCl [pH 7.0]) and washed three times. Approximately 50 μl of suspended RBCs was added to an equal volume of a 2-fold dilution of the peptides in 96-well microtiter plates, followed by incubation for 1 h. The final erythrocyte concentration was 4% (vol/vol). After 1 h, the mixture was centrifuged, and the release of hemoglobin in the supernatant was determined spectrophotometrically at OD₅₄₀. Buffer and 1% Triton X-100 instead of peptides served as a negative control and a positive control, respectively. The percentage of hemolysis was calculated according to the following formula: % hemolysis = (OD_peptide − OD_buffer)/(OD_{Triton X-100} − OD_buffer) · 100.

LPS neutralization assay.

The strength of the peptides to neutralize LPS was assayed by using a commercially available LAL chromogenic kit (QCL 100 Cambrex). The protocol explained in the manufacturer's instructions was strictly followed. The endotoxic principle, LPS in Gram-negative bacteria, activates a proenzyme in the Limulus amebocyte lysate (LAL). This activated enzyme in turn catalytically splits the colored product para-nitroanilide (pNA) from the colorless substrate Ac-Ile-Glu-Ala-Arg-para-nitroanilide and is detected spectrophotometrically at OD₄₁₀. The peptides were dissolved in the pyrogen-free water supplied with the kit, and the pH was adjusted to 7.0 with 1 N HCl or 1 N NaOH (which is prepared in pyrogen-free water). Increasing concentrations of the peptides were incubated with 1.0 endotoxin units (EU) in a total volume of 50 μl for 30 min at 37°C. About 50 μl of LAL reagent was added to the peptide-EU complex and further incubated for 10 min, followed by the addition of 100 μl of substrate. After an incubation of 6 min for the reaction, the release of colored product was recorded at OD₄₁₀. Water in the place of peptides served as a negative control (blank), which was considered 0% inhibition, and the percentage of LPS neutralization was calculated as follows: % LPS neutralization = [(OD_blank – OD_peptide)/OD_blank] · 100.

Fluorescence studies.

All of the fluorescence studies were carried out in a Cary Eclipse fluorescence spectrophotometer (Varian, Inc.) in 10 mM phosphate buffer (pH 7.0) unless otherwise specified.

Aggregation state of the peptides using rhodamine fluorescence.

A 2 μM concentration of the purified rhodamine-labeled peptides was added to increasing concentrations of LPS, and the fluorescence was monitored at an excitation of 485 nm and an emission at 550 to 620 nm in 10 mM phosphate buffer (pH 7.0).

Membrane permeabilization assays.

E. coli BL21(DE3) cells were grown to mid-logarithmic phase in LB broth and diluted to an OD₆₀₀ of 0.5. For outer membrane permeabilization using NPN dye, ∼500 μl of the cells was added to 10 μM NPN dissolved in acetone, and the basal fluorescence was recorded with an excitation of 350 nm and an emission maximum of 420 nm. An increasing concentration of hybrid peptides was added to cells with NPN and fluorescence intensity after the addition of each concentration was recorded. To assess membrane permeability using SYTOX green dye, cells at an OD₆₀₀ of 0.5 were added to 1 μM dye, followed by incubation with shaking at 37°C for 15 min. Basal fluorescence was recorded by excitation at 485 nm and emission at 520 nm, followed by the addition of increasing concentrations of peptides.

Membrane depolarization.

The assay to measure membrane depolarization ability was performed in intact E. coli cells and in spheroplast. For intact cells, the bacteria were grown to mid-log phase, centrifuged, and suspended in 5 mM HEPES and 20 mM glucose (pH 7.4). After a brief wash, the cell pellets were suspended in the same buffer containing 100 mM KCl to an OD₆₀₀ of 0.05. The spheroplast was prepared by suspending the log-phase-grown bacterial pellets in 10 mM Tris–25% sucrose (pH 7.4). The pellets were washed twice and resuspended in the same buffer containing 1 mM EDTA. The cells were incubated for 15 min with shaking and centrifuged. The pellets were immediately dissolved in ice-cold water and further incubated for 10 min at 4°C. The cells devoid of outer membrane, i.e., spheroplasts, were collected by centrifugation, and the pellets were dissolved to an OD₆₀₀ of 0.05 in the same buffer (5 mM HEPES, 20 mM glucose, 100 mM KCl) as intact cells. The depolarization-detecting dye DiS-C₃-5 (3,3′-diethylthiodicarbocyanine iodide) was added to either intact cells or spheroplasts; the basal fluorescence was recorded by exciting the dye at 622 nm, and the emission was collected at 630 to 700 nm. This sample was incubated for approximately 45 to 60 min until the decrease in fluorescence was stable, i.e., the dye partitions into the membrane. This was then followed by the addition of increasing concentrations of different peptides, and the depolarizing ability was recorded as the increase in fluorescence intensity.

Measurement of dissociation of LPS micelles.

The ability of the conjugated peptides to dissociate LPS micelles was studied fluorometrically using FITC-conjugated LPS (FITC-LPS) and also by dynamic light scattering (DLS) measurements. In FITC-LPS, the fluorescence of FITC is self-quenched in LPS micelles, which upon dissociation of micelles would dequench. A basal fluorescence of 0.5 μM FITC-LPS was recorded at an excitation of 480 nm and an emission of 520 nm. The dissociation of LPS micelles was recorded as a function of the increase in fluorescence intensity with the addition of increasing concentrations of peptides. In DLS measurements, the distribution of various sizes of LPS micelles was determined using 0.5 μM LPS. The change in this distribution was observed after the addition of peptides at different LPS/peptide ratios, e.g., 1:0.5, 1:1, and 1:2. The scattering was measured with DLS software provided with the instrument (Brookhaven Instruments Corp., Holtsville, NY), and the scattering data were analyzed using the CONTIN method.

Intrinsic tryptophan fluorescence and acrylamide quenching.

The binding of hybrid peptides to LPS was determined by using tryptophan fluorescence. Tryptophan is an intrinsic fluorescent probe and is extremely sensitive to the polarity of the environment. About 5 μM, the peptide was taken with 500 μl of buffer, and the emission of tryptophan fluorescence was noted in the free state with excitation at 280 nm and emission at 300 to 400 nm. Each peptide sample was titrated with increasing concentrations of LPS, and the fluorescence emission was recorded from 300 to 400 nm. Fluorescence quenching of the tryptophan residue was carried out by adding increasing concentrations of acrylamide from a stock 5 M solution to a solution containing only peptide and also to peptide-LPS (1:4) complexes. The Stern-Volmer constant (K_sv) values were then calculated using F₀/F = 1 + K_sv[Q], where F₀ and F are the fluorescence intensities before and after the addition of quencher and [Q] is the quencher concentration.

CD spectroscopy.

Conformational change of the peptides upon binding to membranes was detected using circular dichroism (CD) spectroscopy. The data were collected using a Chirascan CD spectrometer (Applied Photophysics, Ltd., United Kingdom). The peptide and peptide-micelle complex were scanned from 190- to 240-nm wavelengths in a 0.01-cm path-length cuvette for an average of three scans. Baseline scans were acquired using 10 mM phosphate buffer (pH 7.0). Samples containing only LPS were also acquired with the same settings. An approximately 25 μM concentration of peptides was used with 30 μM LPS to obtain the secondary structure of the peptides. The appropriate baselines were used to subtract the data, and the corrected data were converted to molar ellipticity (degrees·cm² dmol⁻¹).

ITC.

Binding of peptides with LPS micelles were determined with isothermal titration calorimetry (ITC) using a VP-ITC Micro calorimeter (Microcal, Inc., Northampton, MA). Peptides and LPS were dissolved in 10 mM phosphate buffer (pH 7.0) and filtered. LPS at a concentration of 10 μM was loaded into the sample cell, and the reference cell was filled with buffer. The syringe was filled with 1 mM peptide stock. Typically, 25 3.5-μl injections of the peptides were made into the sample cell at 25°C. The sample cell was stirred continuously at 300 rpm. Raw data were collected and fitted using single site binding model in Microcal Origin 5.0 software (Origin Lab Corp., Northampton, MA). Association constant (K_a) and enthalpy change (ΔH) were directly obtained from the software used. ΔG and TΔS were calculated using the fundamental equations of thermodynamics, ΔG = −RTlnK_a and TΔS = (ΔH − ΔG), respectively.

Electron microscopy.

Mid-log-phase grown E. coli cells were incubated with various concentrations of LG21 peptide (3, 8, and 15 μM) and 50 μM LG21R19A for 2 h. The cells were centrifuged, the pellets were dissolved in 10 μl of 10 mM phosphate buffer (pH 7.0), and a drop containing the bacteria was loaded onto carbon-coated electron microscopy grids. The grids were then negatively stained with 2% phosphotungstic acid and examined using a JEOL JEM-1230 electron microscope.

RESULTS AND DISCUSSION

Peptide design.

In previous studies, we have designed de novo a series of 12-amino-acid cationic/hydrophobic peptides with antimicrobial and antiendotoxic activity (56, 57). A sequence motif WKRKRF located at the center of the primary structures of the designed peptide has been demonstrated to be critical for the structure in LPS and activity. An octapeptide or GG8, G-WKRKRF-G, interacted with LPS and adopted boomerang-like conformation in LPS. The amphipathic structure of the boomerang motif is demarcated by close packing of the aromatic side chains of residues W and F, whereas the side chains of the four basic amino acids are distally located. GG8 peptide or the boomerang motif is devoid of antimicrobial and antiendotoxic activities, but its atomic resolution structure in LPS indicated specific interactions. We surmised that the inclusion of the boomerang motif sequence in TA, TB, and KL12-based peptide may abolish LPS induced aggregation, yielding broad-spectrum AMPs (Fig. 1). We have obtained synthetic hybrid peptides of TA, TB, and KL12 containing the boomerang motif at the C terminus (Table 1). Two analog peptides containing Ala replacements are also synthesized to understand role of the β-boomerang motif sequence in activity (Table 1).

FIG 1.

Proposed model of activation of inactive AMPs aggregated in LPS by the β-boomerang motif. The diagram shows the aggregation of AMP in LPS (left panel) and the disruption of LPS outer membrane by nonaggregated AMP upon conjugation with the β-boomerang motif (right panel).

TABLE 1.

Amino acid sequences of the hybrid peptides LG21, FG21, and KG20 and mutated analogs of LG21 with an Ala replacement

Peptide	Amino acid sequencea
LG21	LLPIVGNLLKSLLGWKRKRFG
FG21	FLPLIGRVLSGILGWKRKRFG
KG20	KLLLKLKLKLLKGWKRKRFG
LG21R19A	LLPIVGNLLKSLLGWKRKAFG
LG21W15AF20A	LLPIVGNLLKSLLGAKRKRAG

^aThe amino acid sequences from parent peptides—TB (in LG21), TA (in FG21), and KL12 (in KG20)—are underlined; the β-boomerang motifs are indicated in boldface. The mutation to Ala is italicized in LG21R19A and LG21W15AF20A. All peptides are amidated at their C termini.

Antimicrobial activity and RBC lysis by hybrid peptides.

Table 2 summarizes toxicity of the hybrid peptides LG21, FG21, KG20, and LG21R19A, and LG21W15AF20A against bacterial strains, in MH broth, and RBCs. Remarkably, the hybrid peptides LG21, FG21, and KG20 demonstrate potent antibacterial activity, with low MICs, including Gram-negative strains in comparison to the parent peptides (Table 2). It should be noted that the parent peptides TA, TB, and KL12 were found to be largely ineffective against Gram-negative bacteria (49–51, 58). Interestingly, the mutated analogs of LG21—LG21R19A and LG21W15AF20A—exhibited largely limited bacterial cell killing activity (Table 2). The role of the outer membrane in the bactericidal activity of parent peptides and hybrid peptides correlates well with the membrane depolarization, as studied with the florescent dye DiS-C₃-5, of E. coli cells and spheroplasts, i.e., E. coli cells lacking an outer membrane (49). The hybrid peptides efficiently depolarize E. coli cells and spheroplasts, whereas parent peptides showed less depolarization of E. coli cells and more depolarization of the spheroplasts (see Fig. S1 in the supplemental material). The LG21R19A peptide was least active in depolarizing cells and spheroplasts (see Fig. S1 in the supplemental material). These data indicate that the outer membrane, as a permeability barrier, plays essential roles in the insertion of the peptides.

TABLE 2.

MICs in MH broth and hemolysis of hybrid peptides^a

Organism	MIC (μM)
	Parent peptide			Hybrid peptide or mutant
	TB	TA	KL12	LG21	FG21	KG20	LG21R19A	LG21W15AF20A
Gram-negative bacteria
Escherichia coli (lab strain)	100	>200	50	2	2	4	100	100
Pseudomonas aeruginosa (ATCC 27853)	100	>200	100	4	2	4	200	100
Klebsiella pneumonia (ATCC 13883)	100	>200	>200	2	4	10	200	200
Salmonella enterica (ATCC 14028)	200	>200	>200	2	5	10	>200	>200
Gram-positive bacteria
Bacillus subtilis (lab strain)	25	25	50	2	1	4	50	100
Staphylococcus aureus (ATCC 25923)	25	25	50	2	0.5	4	50	100
Streptococcus pyogenes (ATCC 19615)	25	25	50	2	3	10	100	200
Enterococcus faecalis (ATCC 29212)	50	50	100	5	10	10	100	>200

^aMICs were also determined for the parent peptides TA, TB, and KL12 peptides for comparison. The percent hemolysis values at 100 μM were as follows: LG21, 8.5%; FG21, 9.9%; KG20, 2.4%; LG21R19A, 25%; and LG21W15AF20A, 60%.

In hemolysis assays the hybrid peptides LG21, FG21, and KG20 demonstrated very low activity (Table 2). At a 100 μM peptide concentration, lysis of RBCs has been estimated to be only <10%. However, a somewhat greater hemolysis has been detected for the mutated analogs, the LG21R19A and LG21W15AF20A peptides (Table 2).

We also tested the antibacterial activity of the active hybrid peptides LG21, FG21, and KG20 in LB medium containing 150 mM NaCl. LG21 and FG21 peptides are able to exert antimicrobial activity even in the presence of salt, with MICs ranging from 8 to 10 μM, against test bacteria (Table 3). However, KG20 peptide has been observed to be salt sensitive, yielding rather high MICs (Table 3). A number of AMPs are known to be inhibited under physiological salt concentrations, including β-defensins, magainins, indolicidin, etc. (59–61). Taken together, these results demonstrate that the hybrid peptides LG21, FG21, and KG20 are bestowed with broad-spectrum bactericidal activity and, notably, the activities are retained for LG21 and FG21, even in the presence of salt. Further, it should be noted that TA and TB were poorly hemolytic, whereas KL12 peptide exhibited strong hemolysis, i.e., ∼80%, at 100 μM (49, 53). In other words, LG21 and FG21 retain a low hemolytic activity akin to TB and TA peptides. Further, the inclusion of the boomerang motif in KL12 peptide significantly lowered the hemolytic property of the hybrid KG20 peptide. The lowered hemolytic activity of the KG20 peptide, compared to KL12, may arise due to preferential interactions, as demonstrated in previous studies (56, 57), of the boomerang motif of the hybrid peptide with negatively charged lipids of bacterial membranes over zwitterionic lipids in mammalian cells. The inability of the mutated peptides, LG21R19A and LG21W15AF20A, to exert a bactericidal effect implicates the vital role of the cationic residues and aromatic residues W15 and F20 in the short boomerang motif sequence for the activity of the hybrid peptides. It should be noted that, despite the same cationicity of the LG21W15AF20A peptide akin to LG21, the mutated peptide lacks bactericidal activity. Most strikingly, a single mutation of R19A in LG21R19A peptide also severely reduces the bactericidal activity. The poor bactericidal activity and relatively higher hemolytic activity of the mutated peptides, LG21W15AF20A and LG21R19A, may be related to altered interactions and structures in the context of different cell types. To understand the potential correlation between structure and activity, we utilized the LG21R19A peptide, along with the LG21, FG21, and KG20 peptides, for further studies, as described below.

TABLE 3.

MICs in salt-containing LB medium of hybrid peptides

Organism	MIC (μM)
	LG21	FG21	KG20
Gram-negative bacteria
Escherichia coli (lab strain)	8	10	100
Pseudomonas aeruginosa (ATCC 27853)	8	8	50
Klebsiella pneumonia (ATCC 13883)	8	8	100
Salmonella enterica (ATCC 14028)	8	10	200
Gram-positive bacteria
Bacillus subtilis (lab strain)	10	10	50
Staphylococcus aureus (ATCC 25923)	12.5	10	25
Streptococcus pyogenes (ATCC 19615)	8	12.5	100
Enterococcus faecalis (ATCC 29212)	12.5	10	50

Endotoxin neutralization by hybrid peptides.

LPS or endotoxin neutralization by hybrid peptides has been examined using LAL assay (62). LAL assay is highly sensitive in detecting free LPS at concentrations as low as 1 pM (63). LPS-neutralizing proteins and peptides can sequester LPS, reducing free endotoxin in solution (64, 65). All three hybrid peptides—LG21, FG21, and KG20—demonstrate inhibitory activity in neutralizing LPS (see Fig. S2 in the supplemental material). The LG21 peptide shows a higher potency in endotoxin neutralization than FG21 and KG20 (see Fig. S2 in the supplemental material). LG21 exhibits inhibition of LPS even at 3 μM. At a 10 μM concentration of LG21, >75% inhibition of LPS was detected. Overall, there has been an increase in inhibition with increasing concentrations of LG21, FG21, and KG20 peptides (see Fig. S2 in the supplemental material). The mutated peptide LG21R19A does not display LPS inhibitory activity in a significant way (see Fig. S2 in the supplemental material). Note that a previous study showed that the LPS neutralization ability of TB and TA peptides is highly limited (51). The hybrid peptides LG21, FG21, and KG20 are able to neutralize the toxicity of LPS. It may be worthwhile to consider that the incorporation of the boomerang motif in other AMPs may enhance their antimicrobial and antiendotoxic activities while potentially lowering toxicity.

Self-association of hybrid peptides in LPS.

Rhodamine-labeled peptides are utilized to assess self-associations of hybrid peptides in the presence of LPS. The fluorescence emission intensity of rhodamine is highly sensitive to molecular aggregations whereby aggregations of rhodamine-labeled peptides would show a decrease in fluorescence intensity of the fluorophore due to self-quenching (50). Figure 2 shows a plot indicating differences in fluorescence intensity (ΔF) of rhodamine, measured at emission maxima of 590 nm, for rhodamine-labeled peptides in the absence of LPS and in the presence of LPS at 1, 2, 4, 8, and 10 μM. There has been an increase in fluorescence intensity of rhodamine for LG21, FG21, and KG20 hybrid peptides with increasing concentrations of LPS, demonstrating the plausible absence of aggregations in LPS for these peptides. In contrast, the fluorescence intensity of rhodamine is decreased for LG21R19A peptide, implying aggregation of the mutated peptide in complex with LPS (Fig. 2). Collectively, the aforementioned results suggest that the incorporation of the LPS binding boomerang motif abolishes LPS-induced aggregations in hybrid peptides. The ability to kill Gram-negative bacterial strains by the hybrid peptides correlates well with their lack of aggregations in LPS. The self-association of the LG21R19A peptide in LPS appears to be responsible for its reduced bactericidal activity.

FIG 2.

Association of hybrid peptides with LPS as determined by rhodamine fluorescence. The plot shows the changes in fluorescence intensity, at the emission maxima (ΔF₅₉₀ nm), of rhodamine-labeled LG21 (rhoLG21), FG21 (rhoFG21), KG20 (rhoKG20), and LG21R19A (rhoLG21R19A) peptides as a function of the concentration of LPS. Experiments were performed in 10 mM sodium phosphate buffer (pH 7.0). Concentrations (2 μM) of rhodamine-labeled peptides were individually titrated with increasing concentrations of LPS. The fluorescence was monitored at an excitation of 485 nm and an emission of 550 to 620 nm.

Permeation of E. coli cell membrane and membrane disruption by hybrid peptides.

In order to determine membrane damage caused by the hybrid peptides, we carried out fluorescence studies with E. coli cells, using several membrane probes. NPN and SYTOX green are unable to enter into an intact cell unless the membrane integrity is compromised by the addition of membrane-disrupting compounds. The fluorescence intensity of NPN delineates a drastic increase while bound to membrane lipid components, whereas the fluorescence intensity of the cationic dye SYTOX green enhances in complex with intracellular nucleic acids.

Figure 3 shows the differences in fluorescence intensity (ΔF) of NPN (Fig. 3A) and SYTOX green (Fig. 3B) versus concentrations of hybrid peptides. The fluorescence intensities of NPN and SYTOX green are significantly increased upon the additions of increasing concentrations of LG21, FG21, and KG20 peptides. These results strongly demonstrate that the hybrid peptides LG21, FG21, and KG20 efficiently disrupt the integrity of bacterial outer and inner membranes, causing an influx of the fluorescent molecules NPN and SYTOX green into the cells. Notably, in these assays, there are no significant changes in the fluorescence intensity of these probes upon treatment with inactive LG21R19A peptide (Fig. 3). Further, electron microscopic images were obtained for E. coli cells either in the absence of or after treatment with LG21 peptide at 3, 8, and 15 μM or LG21R19A at 50 μM for 2 h (Fig. 4). The morphology of the cells of the LG21 peptide-treated bacteria is distinctly modified compared to the control (Fig. 4). Plausible membrane or cell wall damage can be seen at a 3 μM concentration (close to the MIC) of LG21 (Fig. 4B) compared to untreated cells (Fig. 4A). At higher concentrations (8 and 15 μM) of LG21, cell shape and integrity changes and ghost-like images are clearly observed (Fig. 4C and D). In contrast, the inactive LG21R19A peptide does not impart any discernible damage to the E. coli cells (Fig. 4E).

FIG 3.

Cell permeabilization by hybrid peptides. (A) Plot showing the changes in fluorescence intensity of NPN at an emission of 420 nm with increasing concentrations of LG21, FG21, KG20, and LG21R19A peptides in the presence of E. coli cells. E. coli cells were incubated with 10 μM NPN, and increasing concentrations of peptides were titrated, in individual experiments, measuring the NPN fluorescence. (B) Plot showing the changes in fluorescence intensity of SYTOX green at an emission of 520 nm with increasing concentrations of LG21, FG21, KG20, and LG21R19A peptides in the presence of E. coli cells. E. coli cells were incubated with 1 μM SYTOX green at 37°C for 15 min. The fluorescence emission of SYTOX green was recorded in the absence of peptides and with increasing concentrations of peptides.

FIG 4.

Cell damage by LG21 in electron microscopy studies. Electron micrographs of negatively stained E. coli cells in the absence (A) or in the presence of 3 μM (B), 8 μM (C), or 15 μM (D) LG21 peptide or 50 μM LG21R19A peptide (E) are shown.

Binding affinity of hybrid peptides with LPS.

In order to better understand the superior activity of hybrid peptides against Gram-negative bacteria, LPS binding parameters were obtained using ITC experiments (see Fig. S3 in the supplemental material). LPS-peptide interactions are endothermic in nature for the active peptides LG21, FG21, and KG20, as disclosed by the upward trend in the ITC heat peaks (see Fig. S3A to C, top panels, in the supplemental material). Usually, entropy-driven or endothermic binding has been observed for LPS-AMP interactions with the gel phase of LPS at 25°C (66, 67). Interestingly, the inactive peptide LG21R19A shows an opposite trend, whereby LPS interactions appear to be exothermic in nature (see Fig. S3D, top, in the supplemental material). The apparent dissociation constant (K_d) values and thermodynamic parameters of LPS-peptide interactions are estimated from the ITC data (Table 4). The hybrid peptides LG21, KG21, and FG20 interacted with LPS with submicromolar affinity with a K_d of 0.6, 0.6, and 0.3 μM, respectively, whereas LG21R19A delineates a comparatively much weaker binding to LPS with a K_d of 10 μM (Table 4).

TABLE 4.

Thermodynamic parameters and binding affinity of the hybrid peptides with LPS as determined from ITC

Peptide-LPS interaction	Ka (μM−1)	ΔH (kcal mol−1)	TΔS (kcal mol−1 degree−1)	ΔG (kcal mol−1)	Kd (μM)
LG21	1.64	0.58	8.9	–8.4	0.6
FG21	1.6	4.4	12.8	–8.4	0.6
KG20	3.2	3.4	12.2	–8.8	0.3
LG21R19A	0.099	–1.7	5.1	–6.8	10

Localization of hybrid peptides in LPS.

We further assessed the insertion of hybrid peptides in LPS micelles using intrinsic tryptophan fluorescence experiments. Fluorescence emission spectra of Trp for LG21, FG21, KG20, and LG21R19A were obtained at various concentrations of LPS (ranging from 1 to 20 μM) (see Fig. S4 in the supplemental material). As seen in the figure, the tryptophan residue has experienced a marked blue shift, i.e., the emission maximum shifted toward a shorter wavelength compared to free peptide, for the LG21, FG21, and KG20 peptides in LPS. In marked contrast, the tryptophan residue of the mutated peptide LG21R19A exhibited a highly limited spectral shift in LPS (Table 5; see Fig. S4 in the supplemental material). The solvent accessibility of the tryptophan residue was judged by fluorescence-quenching studies in the presence of acrylamide either in free solutions or in LPS-containing solutions. The quenching constant (K_sv) values of LG21, FG21, and KG20 peptides in LPS were estimated to be significantly lower in comparison to K_sv values determined in free solution (Table 5). In contrast, the LG21R19A peptide showed comparable K_sv values for the tryptophan residue in LPS and in buffer solution (Table 5). These observations demonstrate that in the active peptides LG21, FG21, and KG20 the tryptophan residue is inserted into a nonpolar environment of LPS with restricted exposure to the solvent, whereas the tryptophan residue of the inactive peptide, LG21R19A, lacks the nonpolar environment of the acyl chains of LPS. In other words, the LG21R19A peptide is predominantly localized at the outer surface or hydrophilic sugar region of LPS with a higher degree of solvent exposure.

TABLE 5.

Emission maximum wavelength (λ_max) and K_sv values of Trp residues of hybrid peptides in buffer (free) and LPS micelles

Peptide	λmax (nm)		Ksv
	Free	LPS	Free	LPS
LG21	358	338	15.6	4.7
FG21	356	338	25.4	7.7
KG20	354	336	27.9	5.8
LG21R19A	356	354	17.9	10.9

Disaggregation of LPS aggregates by hybrid peptides.

High-binding affinity and insertion into the hydrophobic lipid A domain of LPS of the active hybrid peptides may cause structural changes in LPS. We examined the perturbation of LPS using DLS and fluorescence of FITC-labeled LPS. DLS experiments examine the size distribution of LPS aggregates in the presence of peptides (Fig. 5). In free solution, LPS is highly polydispersed, with an average diameter of 806 nm (Fig. 5A). There has been a drastic reduction in the size of LPS aggregates with a concomitant reduction in polydispersion in the presence of LG21 (Fig. 5B), FG21 (Fig. 5C), and KG20 (Fig. 5D) peptides. In contrast, changes in LPS aggregates appear to be largely limited, with an average diameter of 521 nm, in the presence of mutated LG21R19A peptide (Fig. 5E). The average diameters of LPS micelles were estimated for each active peptide to be 85, 87, and 134 nm for LG21, FG21 and KG20, respectively. Hybrid peptide-induced disaggregation of LPS micelles has also been observed based on the fluorescence intensity of FITC in FITC-LPS. The fluorescence intensity of FITC in FITC-LPS is largely quenched in solution due to the aggregation of LPS. Binding of proteins or peptides causing perturbation of LPS-aggregated structures may enhance fluorescence intensity (57, 68, 69). Proteins or peptide-mediated dissociation of LPS have been correlated with their antiendotoxin activity (57, 68, 69). Figure 5F shows the change in fluorescence intensity (ΔF) of FITC-LPS with increasing concentrations of LG21, FG21, KG20, and LG21R19A peptides. There have been drastic increases in ΔF for the active hybrid peptides LG21, FG21, and KG20 in a dose-dependent manner, indicating potential dissociation of LPS aggregates (Fig. 5F). On the other hand, the inactive peptide, LG21R19A, does not cause any detectable dissociation of LPS, as suggested by the lack of increase in the fluorescence intensity of FITC (Fig. 5F).

FIG 5.

Disaggregation of LPS by hybrid peptides. (A to E) Bar diagrams showing the diameter versus the intensity of scattered light for LPS alone (A) or in the presence of LG21 (B), FG21 (C), KG20 (D), or LG21R19A (E). (F) Changes in fluorescence intensity of FITC-labeled LPS as a function of various concentrations of LG21, FG21, KG20, and LG21R19A peptides. All experiments were carried out in 10 mM sodium phosphate buffer (pH 7.0).

Secondary structures of the hybrid peptides.

CD spectroscopy, at the far-UV range (240 to 190 nm), was carried out to assess the global conformations of the hybrid peptides in free solution and in complex with LPS micelles. CD spectra, in free solution, of LG21, FG21, KG20, and LG21R19A delineate a single band at ∼195 nm, indicating random conformations (see Fig. S5 in the supplemental material). CD spectra obtained for the hybrid peptides and mutant analog in LPS micelles are characterized by two negative bands at ∼220 to 225 nm and at ∼208 to 210 nm, indicating predominantly helical conformations. Intriguingly, the active hybrid peptides LG21, FG21, and KG20, as well as the inactive mutant LG21R19A, assume helical conformations in LPS. Also, the TA, TB, and KL peptides demonstrated helical conformations in LPS and random conformations in water (49, 51). In other words, irrespective of the activity, interactions of these peptides with LPS induce conformational transitions into helical states. At present, it is not clear from the CD studies whether the LPS binding motif assumes the boomerang-like conformations in LPS micelles. Atomic resolution structures of these hybrid peptides and the mutated peptides in complex with LPS micelles are required in order to determine specific structural features.

Conclusion.

The lower activity of AMPs toward Gram-negative bacteria may arise from their self-association in LPS outer membrane. In the present study, we demonstrated that an LPS-binding peptide motif when conjugated with inactive AMPs temporins and KL12 abolished LPS-induced aggregations, yielding broad-spectrum salt-resistant hybrid AMPs. Hybrid peptides demonstrate neutralization of endotoxin and limited hemolysis. As a mode of action, hybrid peptides bind LPS with high affinity and disrupt the higher-order structures of LPS. These traits of the hybrid peptides are correlated with the efficient disruption of the outer membrane and cell permeabilization. The boomerang motif is critically involved in the broad-spectrum activity of the hybrid peptides since a single mutation LG21R19A in LG21 produced an inactive peptide that shows self-association in LPS. The lack of bactericidal and endotoxin neutralization activity of the LG21R19A peptide stems from its low-affinity binding to LPS micelles and limited perturbation of LPS structural states. We surmise that the hybrid peptides obtained here and their mode of action through the LPS outer membrane would be useful for designing a new class of cell wall-permeabilizing AMPs. Such AMPs can be tested further for in vivo efficacy in animal models.