The effects of LPS on the activity of Trp-containing antimicrobial peptides against Gram-negative bacteria and endotoxin neutralization.

Acta Biomaterialia xxx (2016) xxx–xxx

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The effects of LPS on the activity of Trp-containing antimicrobial peptides against Gram-negative bacteria and endotoxin neutralization Dejing Shang a,b,⇑, Qian Zhang a, Weibing Dong a,b, Hao Liang a, Xiaonan Bi a a b

Faculty of Life Science, Liaoning Normal University, Dalian 116081, China Liaoning Provincial Key Laboratory of Biotechnology and Drug Discovery, Dalian 116081, China

a r t i c l e

i n f o

Article history: Received 16 September 2015 Received in revised form 11 January 2016 Accepted 18 January 2016 Available online xxxx Keywords: Trp-containing peptides Outer membrane Permeability Disassociation Antiendotoxin

a b s t r a c t A series of synthesized Trp-containing antimicrobial peptides showed significantly different antimicrobial activity against Gram-negative bacteria despite having similar components and amino acid sequences and the same net positive charge and hydrophobicity. Lipopolysaccharide (LPS) in the outer membrane is a permeability barrier to prevent antimicrobial peptides from crossing into Gramnegative bacteria. We investigated the interaction of five Trp-containing peptides, I1W, I4W, L5W, L11W and L12W, with LPS using circular dichroism (CD), IR spectroscopy, isothermal titration calorimetry (ITC), dynamic light scattering (DLS), zeta-potential measurements and confocal laser scanning microscopy, to address whether bacterial LPS is responsible for the different susceptibilities of Gram-negative bacteria to Trp-containing peptides. Our data indicate that I1W and I4W penetrated the LPS layer and killed Gram-negative bacteria by a ‘‘self-promoted uptake” pathway in which the peptides first approach LPS by electrostatic forces and then dissociate LPS micelle. This process results in disorganization of the LPS leaflet and promotes the ability of the peptide to cross the outer membrane into the inner membrane and disrupt the cytoplasmic membrane. Although L5W, L11W and L12W strongly bind to LPS bilayers and depolarize bacterial cytoplasmic membranes, similar to I1W and I4W, they are unable to destabilize LPS aggregates and traverse through the tightly packed LPS molecules. This study increases our understanding of the mechanism of action of these peptides in the LPS outer membrane and will help in the development of a potent broad-spectrum antibiotic for future therapeutic purposes. Statement of Significance Tryptophan (Trp) residues show a strong preference for the interfacial region of biological membranes, and this property endows Trp-containing peptides with the unique ability to interact with the surface of bacterial cell membranes. In this manuscript, we report the membrane interaction of Trp-containing peptide to address whether bacterial LPS is responsible for the different susceptibilities of Gramnegative bacteria to Trp-containing peptides. Based on the data collected, we propose a molecular mechanism for the peptide-LPS interactions that allows the peptides to traverse or prevents them from transversing the LPS layer and the target inner membrane. The data should help in the development of a potent broad-spectrum antibiotic for future therapeutic purposes. Ó 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction The dramatic increase in multiple-drug-resistant pathogenic microorganisms over the past two to three decades represents a serious challenge to human health and medicine [1,2]. Multiple- drug-resistant strains of particular concern include ⇑ Corresponding author at: Liaoning Provincial Key Laboratory of Biotechnology and Drug Discovery, Liaoning Normal University, Dalian 116081, China. E-mail address: [email protected] (D. Shang).

Gram-negative bacteria such as Pseudomonas aeruginosa, which is associated with infections of chronic and traumatic wounds, burns, and medical implants, and Entero bacteriaceae (including Escherichia coli and Klebsiella pneumoniae), which is associated with infections in hospitals and other healthcare facilities [3–5]. This genetic variability of pathogens and the growing multi-drug resistance of bacterial strains have stimulated an intensive research effort to develop new antibiotics that exhibit novel mechanisms of action. Antimicrobial peptides (AMPs) are promising candidates for the development of new antimicrobial agents due to their

http://dx.doi.org/10.1016/j.actbio.2016.01.019 1742-7061/Ó 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: D. Shang et al., The effects of LPS on the activity of Trp-containing antimicrobial peptides against Gram-negative bacteria and endotoxin neutralization, Acta Biomater. (2016), http://dx.doi.org/10.1016/j.actbio.2016.01.019

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D. Shang et al. / Acta Biomaterialia xxx (2016) xxx–xxx

unusually broad spectrum of activity and the low level of associated induced resistance [6–8]. In addition, some AMPs are also recognized as a possible source of pharmaceuticals for the treatment of septic shock syndromes caused by the lipopolysaccharide (LPS) of Gram-negative bacteria [9]. It is generally known that most AMPs target the cell membrane, so the ability of AMPs to interact with the membrane affects their antimicrobial activity. AMPs containing tryptophan (Trp) residues display more potent antimicrobial activity than those with either phenylalanine or tyrosine [10,11]. The bulkier Trp residues reportedly exhibit the unique property of being able to interact with the interfacial region of a membrane, allowing the peptides to partition into the bilayer interface [12,13]. We have previously reported the in vitro activity of a series of Trp-containing peptides that were designed and synthesized by substituting the isoleucine or leucine residues at sites Ile1, Ile4, Leu5, Leu11 and Leu12 with Trp residues based on the structure of L-K6, a peptide derived from the frog skin peptide temporin-1CEb [14,15]. Among the 11 designed peptides, L5W, L11W and L12W exhibited decreased or no antimicrobial activity against Gram-negative bacteria, particularly against Enterobacter cloacae, K. pneumonia and Proteus mirabilis [16,17]. For most AMPs that are not active against Gram-negative bacteria, the outer membrane is believed to be a major barrier that modulates the transport of AMPs across the bacteria cell membranes [18]. LPS present in the outer leaflet of the Gram-negative bacteria cell membrane is generally thought to be a protective wall that renders bacteria resistant to a variety of host defense molecules. Moreover, LPS, also known as endotoxin, can cause an up-regulation of proinflammatory cytokine production and result in septic shock syndromes in human [19,20]. Some studies have shown that LPS actively regulates the membrane insertion and antibacterial activities of many AMPs [21] through ionic interactions that lead to peptide ‘‘self-promoted” uptake; upon interaction, the peptides fold, intercalate into the phospholipid bilayer and exert their antimicrobial activities [18,22–24]. And AMPs with LPS binding and neutralizing ability may be considered to be a possible therapeutic target in patients. However, the details of the interactions of the peptides with LPS are still not fully known. In this present study, we synthesized five Trp-containing peptides, I1W, I4W, L5W, L11W and L12W, and investigated the interaction of these peptides with LPS using various biophysical and biochemical methods, including circular dichroism (CD), IR spectroscopy, isothermal titration calorimetry (ITC), dynamic light scattering (DLS), zeta-potential measurements and laser confocal microscopy, to address whether bacterial LPS is responsible for the different susceptibilities of Gram-negative bacteria to different peptides. Based on the data collected, we propose a molecular mechanism for peptide-LPS interactions that allows the peptides to traverse or prevents them from transversing the LPS layer and targeting the inner membrane. Moreover, five Trp-containing peptides can inhibit the LPS-induced proinflammatory response through direct binding to LPS.

2. Materials and methods 2.1. Bacterial strains The following bacterial strains were acquired from the China General Microbiological Culture Collection Centre: E. coli (AS1.349), P. aeruginosa (CGMCC1.860), E. cloacae (CGMCC 1.58), and K. pneumoniae subsp. pneumoniae (CGMCC1.176). The bacterial strain P. mirabilis (CICC22931) was acquired from the China Centre for Industrial Culture Collection.

2.2. Synthesis of peptides The peptides used in this study, I1W, I4W, L5W, L11W and L12W, were synthesized in crude form by GL Biochemistry (Shanghai, China) using a standard Fmoc solid-phase peptide synthesis protocol. The peptides were purified to near homogeneity (>95%) using reverse-phase high-performance liquid chromatography (HPLC) on a 2.2 25 cm Vydac 218TP1022 (C-18) column equilibrated with aqueous acetonitrile/trifluoroacetic acid (0.1%). Purified peptides were analyzed directly with HPLC–coupled electrospray ionization tandem mass spectrometry or with matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (Fig. S1). 2.3. Confocal laser scanning microscopy Membrane permeability assays were performed using a vital staining probe mixture (LIVE/DEADÒ BacLightTM Bacterial Viability kit; Invitrogen) according to the manufacturer’s instructions. E. coli cells were grown to late exponential phase and washed three times with phosphate-buffered saline. Bacterial cells suspended in PBS buffer were then treated with the Trp-containing peptides at a final concentration of 1 MIC (I1W: 3.13 lM; I4W: 6.25 lM; L5W: 2.50 lM; L11W: 2.50 lM; and L12W: 9.37 lM) for 1.5 h at 37 °C. The two nucleic acid stains provided in the commercial kit, SYTO9 and propidium iodine (PI), were added to the treated cells and stained for 30 min in the dark. The stained cells were imaged using confocal laser scanning microscopy (LSM 710, Carl Zeiss, Germany) with excitation/emission wavelengths of 480/500 nm and 490/635 nm for SYTO9 and PI, respectively. Live bacteria were stained with the SYTO 9 green fluorescent nucleic acid stain, and dead bacteria were stained with the PI red fluorescent dye. 2.4. Outer membrane permeabilization assay Outer membrane permeability was analyzed by 1-N-phenylnaph-thylamine (NPN) dye uptake [25]. Briefly, bacterial cells were grown to mid-log phase and resuspended in PBS buffer to an OD600 of 0.5. A final concentration of 10 lM NPN was added to 500 ll of the bacterial cells, and after an increasing concentration of peptides from 0 to 16 lM was added to cells with NPN, the basal fluorescence intensity was recorded with an excitation of 350 nm and an emission maximum of 420 nm. 2.5. Preparation of bacterial spheroplasts Spheroplasts of the tested Gram-negative bacteria were prepared using the osmotic shock procedure as previously described [26]. Briefly, bacteria grown in mid-log phase were harvested and washed twice with 10 mM PB buffer. Subsequently, the cells were resuspended in 0.5 M sucrose solution in PB, and lysozyme (to a final concentration of 80 lg/ml) was added to the cell suspension for 2 h at 37 °C with rotary mixing. The spheroplasts were collected by centrifugation and resuspended in a buffer containing 5 mM HEPES, 20 mM glucose and 100 mM KCl, pH 7, for analysis. Additional MIC experiments were performed exactly as described for the intact bacteria [26]. 2.6. Preparation of LPS mixed small and large unilamellar vesicles (SUVs and LUVs) Phosphatidylcholine (PC) and phosphatidylglycerol (PG) were purchased from Sigma (Shanghai, China). SUVs were prepared by sonication of the required amount of PG/PC (1:3, w/w)



or PG/PC/LPS (1:3:4, w/w/w). The phospholipids were dissolved in chloroform at each of the previously described ratios and dried, and the solvents were removed via rotary evaporation to form a multilamellar liposome. Any remaining trace amounts of organic solvent were then completely removed by lyophilization overnight, and the dry lipid film was resuspended with gentle vortexing in 2–3 ml of 5 mM N-Tris (hydroxymethyl) methyl-2aminoethanesulfonic acid (TES) buffer (pH 7.4) containing 0.1 M NaCl. The suspension was sonicated at 40 °C for 8 min using a probe-type sonicator. The SUVs were immediately used for CD measurements. LUVs with encapsulated calcein were prepared using the freeze–thaw method [16]. Briefly, the phospholipids were dissolved in chloroform at each of the previously described ratios. After vacuum evaporation and overnight drying, a dye solution (90 mM calcein, 20 mM TES, 100 mM NaCl, pH 7.4) was added to each sample. LUVs were prepared via ten freeze–thaw cycles in liquid nitrogen followed by incubation in a water bath at 50 °C. The suspensions were then extruded ten times through 200-nm polycarbonate membranes. After extrusion, the calcein-labeled vesicles were separated from free calcein via gel filtration through a Sephadex G-50 column and eluted using TES buffer. The calceinlabeled LUVs (90 lM final concentration) were used for leakage measurements. 2.7. Leakage of calcein from liposomes containing LPS The ability of the peptides to induce calcein leakage from LUVs containing LPS was measured. LUVs were incubated for 30 min in a suspension containing various concentrations of peptides (0.78–25 lM). The fluorescence intensity of the calcein released from the LUVs was recorded at 530 nm with an excitation wavelength of 493 nm. Complete (100%) release of calcein was induced by the addition of 0.1% Triton X-100. All experiments were conducted at room temperature, and measurements were repeated three times under each condition. The percentage of released calcein induced by the peptides was calculated using the following equation: Release (%) = 100 (F F0)/(Ft F0), where F and Ft are the fluorescence intensities before and after the addition of Triton X-100, respectively, and F0 is the fluorescence intensity of the intact vesicles. 2.8. Bacteria net surface negative charges Fluorescein isothiocyanate labeled poly-l-lysine (FITC-PLL) was used to determine the relative net surface negative charges of the bacteria [27]. Bacterial cells were incubated overnight at 37 °C in Mueller–Hinton (MH) broth and washed with HEPES buffer. Subsequently, 50 lL of inoculum (105 CFU/mL) of a log-phase bacterial culture was mixed with 50 lL FITC-PLL (10 lg/ml) and then incubated at 37 °C for 10 min. The amount of FITC-PLL remaining in the supernatant was determined fluorometrically (excitation at 500 nm and emission at 530 nm) (Varioskan Flash, Thermo Scientific, Beijing) with and without bacterial exposure. A lower amount of unbound FITC-PLL indicated a smaller PLL repulsion and more negatively charged cell envelope. 2.9. Zeta potential Zeta-potential measurements were performed on a Zetasizer Nano ZS (Malvern Instruments, UK) equipped with a 633 nm HeNe laser. Bacterial cells grown at mid-log phase were diluted in 10 mM sodium phosphate buffer (pH 6.8) to a final OD600 of 0.2, loaded into a disposable zeta cell with a gold electrode and allowed to equilibrate for 3 min at 25 °C. Increasing concentrations of peptides were then added to the cells, and the measurements were

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taken. A total of 5 measurements of 100 runs each was performed for all the concentrations of peptide. 2.10. ITC ITC was performed using a MicroCal ITC 200 (GE instrument Co., Beijing, China). Solutions were degassed under a vacuum prior to use. Peptide aliquots (2 ll at 0.5 mM) dissolved in 50 mM phosphate buffer (pH 6.8) were titrated every 2 min into the sample cell filled with 200 ll of 0.05 mM LPS in 50 mM phosphate buffer (pH 6.8) under constant stirring at 37 °C for 30 injections. The heat of the interaction measured by the ITC instrument after each injection was plotted against time. The heats of dilution were determined in control experiments by injecting either peptide solution or lipid suspension into buffer. The heats of dilution were subtracted from the heats determined in the corresponding peptide-lipid binding experiments. The total heat signal from each experiment was determined as the area under the respective single peaks and plotted against the [peptide]:[lipid] molar ratio. The experiment was performed in triplicate. 2.11. Dissociation of LPS aggregates The dissociation of LPS micelles by peptides was studied fluorometrically using FITC-conjugated LPS (FITC-LPS). FITC-LPS (0.5 lg/ml) was treated with increasing concentrations of peptides (2, 4, 8, 16, 32, 64, and 128 lM). The basal fluorescence of FITC-LPS as a function of the change in the aggregation state of LPS in 10 mM sodium phosphate buffer (pH 6.9) was monitored at an excitation of 480 nm and an emission of 515 nm using a Varioskan Flash Microplate Reader (Thermo Scientific Co., Beijing). The fluorescence data of both sodium phosphate buffer and peptides alone at 515 nm were taken for background subtractions. Dissociation of the aggregated state of FITC-LPS was measured as an increase in its fluorescence. 2.12. DLS studies Dissociation of LPS micelles by peptides was also studied by DLS measurements performed on a Zetasizer Nano ZS (Malvern Instruments, UK) equipped with a 633 nm HeNe laser. First, LPS aggregates were prepared. LPS was solubilized in 10 mM sodium phosphate buffer (pH 6.9), extensively vortexed, sonicated at 60 °C for 30 min, and subjected to 3–4 temperature cycles between 20 and 60 °C. Finally, the lipid suspension was incubated at 4°C for atleast 12 h before use. For DLS measurements, a final concentration of 1 lM LPS was dissolved in 10 mM sodium phosphate buffer (pH 6.0), and the distribution of various sizes of LPS micelles in the presence of 2 lM peptides and in the absence of peptides was determined. The scattering data were analyzed using the CONTIN method provided with the instrument. 2.13. CD spectroscopy To study the propensity of the peptides to self-assemble in LPS and PBS buffer environments, the CD spectrum of the peptides was measured with a Jasco-810 spectropolarimeter (JASCO, Victoria, B.C., Canada). The spectrum was scanned at 25 °C in a capped quartz optical cell with a path length of 1 mm in the wavelength range of 190 and 300 nm at 0.5-nm intervals with a scan rate of 20 nm/min. The peptide concentration for CD measurement was 0.25 mg/ml in 10 mM PBS buffer pH 7.4 or in the presence of 0.1% (0.22 mM) LPS. Mean residue ellipticities were expressed as [h] (degrees cm2 /dmol). The spectra of three consecutive scans were averaged, and the CD spectra of PBS buffer or LPS solutions without the peptide were used as baseline spectra. The CD spectra


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of the appropriate solvent were subtracted from each corresponding peptide spectrum. The percentage of helicity was estimated with the program CDNN.

were determined by enzyme-linked immunosorbent assay (ELISA) (Rapidbio, Shanghai, China). All assays were performed in duplicate.

2.14. IR experiments

3. Results

IR spectra were recorded using a Mettler-Toledo ReactIR 15 system equipped with an MCT detector and a Dsub AgX SiComp in situ probe. For amide I region measurements (from 1700 to 1600 cm1), 2 mg of LPS was dissolved in 0.5 ml of D2O, and a spectrum was collected at a resolution of 4 cm1 for a total of 256 scans and used as the background. Then, 1 mg of peptide was added to the solution, and spectra were collected after 1 h. To resolve overlapping bands, we processed the spectra using origin software. Second-derivation spectra were calculated to identify the positions of the component bands in the spectra. These wave numbers were used as the initial parameters for curve fitting with Gaussian component peaks. The relative contents of the different secondary structure elements were estimated by dividing the areas of the individual peaks assigned to a specific secondary structure by the total area of the resulting amide I band.

To correlate antimicrobial activity with binding to and permeation of bacterial LPS, we used five Trp-containing antimicrobial peptides, I1W, I4W, L5W, L11W and L12W, which are active against Gram-positive bacteria; however, L5W, L11W and L12W are inactive against the Gram-negative bacteria E. cloacae, K.pneumoniae and P. mirabilis. The sequence of the five peptides and their antibacterial activity are given in Table 1. These peptides have similar amino acid compositions (I1W and I4W: 6 K, 1W, 1S, 3L and 2I; L5W, L11W and L12W: 6 K, 1W, 1S, 2L and 3I) and sequences and the same chain length, net positive charge and hydrophobicity, but the Trp residues are located at sites 1, 4, 5, 11 and 12 from the amino terminal in I1W, I4W, L5W, L11W and L12W, respectively.

2.15. Cell culture and viability detection by methyl thiazolyl tetrazolium (MTT) assay

To understand whether the peptides disrupt the outer membrane of bacterial cells, we studied the outer membrane permeability of Gram-negative bacteria using a hydrophobic fluorescence probe, 1-N-phenylnapthylamine (NPN). The fluorescence of NPN is quenched when exposed to an aqueous environment, but when the outer membrane is disturbed, the dye can enter the hydrophobic environment and increase in fluorescence intensity. As shown in Fig. 1A–D, the addition of I1W and I4W induced a significant increase in the fluorescence intensity of NPN in the four Gram-negative bacteria in a concentrationdependent manner. At approximately 2–8 lM of the peptides, the fluorescence intensity reached a plateau, which indicated that I1W and I4W can permeabilize the LPS outer membrane, neutralizing the toxicity of LPS and disrupting the integrity of the outer membrane. Comparatively, in the presence of L5W, L11W and L12W, the fluorescence intensity of NPN slightly increased in E. coli cells in a concentration-dependent manner (Fig. 1A), but the three peptides were unable to induce NPN uptake in E. cloacae, K. pneumoniae and P. mirabilis cells (Fig. 1B–D), suggesting that the outer membrane of Gram-negative bacteria completely prevented L5W, L11W and L12W from entering E. cloacae, K. pneumoniae and P. mirabilis cells. This result is consistent with the antimicrobial activity of these peptides against Gram-negative bacteria previously detected in our laboratory [16,17]. To mimic the effect of the Trp-containing peptides on the outer membrane permeability of Gram-negative bacteria, LUVs containing LPS composed of LPS/PC/PG at a 4/3/1 M ratio were chosen as the model membrane, and LUVs without LPS (PC/PG: 3/1) were chosen as the control. Calcein leakage was used to evaluate the disruption of the membrane structure by the peptides. As shown in

The murine macrophage cell line RAW 264.7 was obtained from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee (Shanghai, China). RAW 264.7 cells were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum. Cells were subcultured at a density of 105/well in 6well dishes for 18 h and then washed with fresh medium before stimulation. Cell viability was determined using MTT assays. Cells were seeded at 5 103 cells/well in a 96-well plate 24 h before peptide treatment. Cells were either treated with various concentrations of peptides or without peptides. Following incubations with peptides of 1, 6 or 24 h, 10 ll of 5 mg/ml MTT solution was added to each well and incubated for 4 h. The purple–blue MTT formazan precipitate was dissolved in 150 ll of DMSO. The absorbance was determined using a microplate reader at 490 nm. Experiments were run in triplicate, and results are expressed as a percentage of the inhibition for viable cells. 2.16. Cytokine assays RAW 264.7 cells were plated at a density of 106/well in 24-well plates, incubated overnight to permit adherence, and then washed with fresh medium before stimulation. The cells were stimulated with LPS (100 lg/ml) and then treated with the concentrations at 5 lM (I1W, I4W and L5W) or 20 lM (L11W and L12W) peptides for 24 h. In all cases, the supernatants of the RAW 264.7 cultures were collected, and the concentrations of tumour necrosis factora (TNF-a) and interleukin-6 (IL-6) in the RAW 264.7 supernatants

3.1. The effects of peptides on the outer membrane permeability of Gram-negative bacteria

Table 1 Minimum inhibitory concentration of peptides against the spheroplasts of tested bacteria. Peptides (Amino acid sequence)

MIC (lM) of spheroplasts /intact bacteria* E. coli

K. pneumoniae

E. cloacae

P. mirabilis

I1W (WKKILSKIKKLLK-NH2) I4W (IKKWLSKIKKLLK-NH2) L5W(IKKIWSKIKKLLK-NH2) L11W(IKKILSKIKKWLK-NH2) L12W(IKKILSKIKKLWK-NH2)

3.13/3.13 6.25/6.25 1.56/2.50 1.56/2.50 6.25/9.37

25.0/25.0 25.0/25.0 25.0/>100 50.0/>100 50.0/>100

12.5/12.5 12.5/12.5 12.5/>100 50.0/>100 25.0/>100

6.25/6.25 12.5/12.5 12.5/>100 50.0/>100 50.0/>100

* MIC: minimal peptide concentration required for total inhibition of the viability of intact cells and spheroplasts of Gram-negative bacterial cells. The values are the means of three independent experiments performed in four replicates.



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Fig. 1. Peptide-induced permeability of the outer membrane in E. coli (A), K. pneumoniae (B), E. cloacae (C) and P. Mirabilis (D). Bacterial cells were incubated with NPN in the presence of various concentrations of peptides. The fluorescence intensity was measured when NPN was inserted into the hydrophobic interior of the outer membrane. (E) and (F) calcein release caused by peptides from LPS/PC/PG and PC/PG liposomes, respectively. Each value represents the mean of three independent experiments.

Fig. 1E, when LPS was present in the membrane, I1W and I4W caused evident calcein leakage from the negatively charged membranes of LPS/PC/PG liposomes, and the release rate was 100% at a molar ratio (peptide/lipids) greater than 3, the leakage effect induced by L5W, L11W and L12W was considerably decreased, with the largest release rate only 50% at molar ratios (peptide/ lipids) greater than 6. However, when LPS was absent from the membrane, L5W, L11W and L12W, much like I1W and I4W, could cause calcein leakage from the PC/PG liposomes, and the release rate was 100% at molar ratios (peptide/lipids) greater than 3 (Fig. 1F). This result could be related to the presence of LPS, which is the main component of the outer membrane of Gram-negative bacteria and may have affected the permeabilization of bacterial membranes by L5W, L11W and L12W. Furthermore, assessment of the antimicrobial activity of the peptides in intact cells and spheroplasts of Gram-negative bacteria showed an effect of the outer LPS membrane on AMP activity. As shown in Table 1, I1W and I4W exhibited similar bactericidal activity for spheroplasts and intact cells of Gram-negative bacteria, but L5W, L11W and L12W had a significant increase in bactericidal

activity against spheroplasts of Gram-negative bacteria compared with intact cells, particularly against spheroplasts of E. cloacae, K. pneumoniae and P. mirabilis cells. The MIC values decreased 2– 8-fold. 3.2. The effects of peptides on the inner membrane permeability of E. coli cells The depolarization level of the cell membrane represents inner membrane permeability. DiSC3 [5] is a membrane potentialsensitive dye. The fluorescent dye exhibits decreased fluorescence when it accumulates in healthy polarized cell membranes, but a peptide that depolarizes the membrane electrical potential can lead to the release of DiSC3 [5] from the membrane and an increase in fluorescence [28]. Data depicting the depolarization of the bacterial inner membranes in spheroplasts and intact cells of E. coli are shown in Fig. 2A–E. The results demonstrated that I1W and I4W induced similar levels of membrane depolarization in intact E. coli cells and spheroplasts, but L5W, L11W and L12W at a concentration of 1 MIC significantly induced membrane depolariza-


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Fig. 2. Cell membrane depolarization in E. coli spheroplasts and intact E. coli cells induced by the peptides I1W (A), I4W (B), L5W (C), L11W (D) and L12W (E) at different concentrations. The depolarization in intact S. aureus cells was used as a positive control. The peptides were added to the spheroplasts or intact bacterial cells that were preequilibrated with the membrane potential fluorescent probe 3, 30 -dipropylthiadicarbocyanine iodide (DiSC3) for 60 min. Fluorescence recovery was recorded. Each value represents the mean of three independent experiments.

Fig. 3. Confocal laser scanning microscopy images of E. coli. Dead cells were stained red and live cells were stained green using the LIVE/DEADÒBacLightTM Bacterial Viability kit. The fluorescence emission of SYTOX 9 (green) and PI (red) was recorded in the untreated control group and in cells treated with the peptides at concentrations of 1 MIC (I1W: 3.13 lM; I4W: 6.25 lM; L5W: 2.50 lM; L11W: 2.50 lM; and L12W: 9.37 lM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

tion in E. coli spheroplasts and had little effect on the membrane charge of intact E. coli cells. The permeability of the cytoplasmic membrane was visualized after staining with two fluorescent nucleic acid stains, SYTO 9 and PI. Viable cells with intact membranes appear green when the membrane-permeable stain SYTO 9 enters the cells. E. coli cells with compromised membranes are stained red by PI, a membraneimpermeant stain. Untreated E. coli cells were only stained green (live); however, compared with the non-treated control, red (dead) signals could be clearly detected when bacterial cells were treated with peptides at the concentration of 1 MIC for 60 min (Fig. 3), suggesting that the increased membrane permeability resulted in the entrance of PI into Gram-negative cells, as indicated by the decreased intensity ratio of SYTO/PI. The five peptides at the concentration of 1 MIC exhibited similar disruption abilities to the inner membrane of E. coli cells. 3.3. Surface charge neutralization of bacterial cells by peptides The binding of positively charged PLL to the negatively charged surface of bacteria results in a decrease in the fluorescence of FITC.

Thus, there was an inverse relationship between the amount of cell surface charge and the fluorescence intensity. The relative surface charge values of four Gram-negative bacteria are presented in Fig. 4A. There were negative charges on the surfaces of all strains. As expected, PLL binding was observed to be higher in E. coli strains than in the other three Gram-negative bacteria. There was no significant difference in surface charge between K. pneumoniae, E. cloacae and P. mirabilis. The zeta potential, an indicator of accessible surface charges, was measured for Gram-negative bacteria cells in the presence of increasing amounts of peptides. The LPS outer membrane of Gram-negative bacteria is known to contribute to a negative zeta potential. Our results showed that the four Gram-negative bacteria have high negative surface charges corresponding to negative zeta potential values of approximately 35 to 40 mV (Fig. 4B–E). Upon the addition of the peptides I1W, I4W, L5W, L11W and L12W at concentrations of 0–300 lM to the different Gramnegative bacterial cells, the zeta potential dramatically increased towards positive values, suggesting that surface charge compensation occurred between bacterial cells and all peptides (Fig.4B–E). At higher peptide concentrations (>150 lM), overcompensation of



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Fig. 4. (A) Net surface charge for four Gram-negative bacteria based on FITC-PLL binding. A log-phase bacterial culture (105 CFU/ml) was mixed with FITC-PLL (10 lg/ml) and then incubated at 37 °C for 10 min. The amount of FITC-PLL remaining in the supernatant was determined fluorometrically (excitation at 500 nm and emission at 530 nm) with and without bacterial exposure. A lower amount of unbound FITC-PLL corresponds to a smaller PLL repulsion and more negatively charged cell envelope. Data are expressed as the mean ± SE of 6 independent tests.**p < 0.01. (B)–(E) Changes in the zeta potential of E. coli, K. pneumoniae, E. cloacae and P. Mirabilis cell membranes in the presence of the peptides I1W, I4W, L5W, L11W and L12W.

surface charge by approximately +0.22 to +2.52 was observed only for E. coli cells and all of the peptides (Fig. 4B). When the peptide concentrations were increased to 300 lM, an overcompensation of surface charge was observed for the suspension of I1W and the other three Gram-negative bacteria, K. pneumoniae, E. cloacae and P. mirabilis. Similar results were observed among I4W and E. cloacae and P. mirabilis (Fig.4C–E). However, overcompensation was not observed in theother three bacteria with L5W, L11W and L12W. Such charge overcompensation indicates that the Trpcontaining peptides I1W or I4W not only bind to the surface of the outer membrane of Gram-negative bacterial cells (except for the suspension of I4W and K. pneumoniae) and neutralize all charges but also insert slightly into the outer membrane. 3.4. The binding of peptides and LPS To better understand the increased activity of the peptides against Gram-negative bacteria, the enthalpy change caused by the binding of the peptides to LPS was determined using ITC experiments by titrating the peptide into the LPS at 37 °C. All titrations for LPS and the peptides indicated an exothermic process with negative enthalpy changes (4Hc) after each titration step (Fig. 5A–E), and binding saturations were observed at the highest molar ratio for L11W and L12W (peptide:LPS = 3.5) after 40 min and the lowest molar ratio for I1W and I4W (peptide:LPS = 1.5) after 20 min. The titrations of I1W and I4W indicated a two-step process: the downward trend of the ITC profiles indicated an exothermic process in the first 25 min, followed by an upward trend indicating an endothermic reaction. The thermodynamic parameters of the LPS-peptide interactions were estimated from the ITC data

(Table 2). The maximum enthalpy changes 4Hc of the peptides binding to LPS indicated relatively low values of 5 to 8 kJ/mol. The dissociation constants (Kd) of I1W and I4W were estimated to be 0.29 lM and 0.37 lM, respectively, and the dissociation constants of L11W and L12W were 4.76 lM and 1.67 lM, suggesting that I1W and I4W had much stronger interaction than L11W and L12W. As the binding of the peptide to LPS occurs concomitantly with folding, the CD spectra were analyzed to assess the global conformations of the peptide in free solution and in complex with LPS micelles. As shown in Fig. 6A, the Trp-containing antimicrobial peptides showed an unordered conformation in PBS solution but exhibited conformational changes in LPS micelles, with two negative bands at 208 to 210 nm and at 220 to 225 nm, indicating predominantly helical conformations (Fig. 6B). Using the CONTIN method provided with the instrument, the calculated helical contents for I1W, I4W, L5W, L11W and L12Wwere 85.93%, 84.57%, 70.81%, 59.34% and 46.38% in LPS micelles, respectively, and 2.92–7.75% in PBS solution. The results suggested that irrespective of their activity, these peptides had a conformational transition into a helical structure in negatively charged environments due to electrostatic interactions between the positively charged residues in the peptide and the negatively charged lipids. Further secondary structure data for these Trp-containing peptides in LPS micelles were obtained from FTIR studies. The amide I region spectra, as well as the fitted band components of the peptides bound to LPS, are shown in Fig. 6C. Assignment of the different secondary structures to the various amide I regions was calculated according to the values previously used [29,30]. The data indicate a strong band at approximately 1654 cm1 in I1W, 1651 cm1 in I4W,


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Fig. 5. Binding of peptides (A) I1W, (B) I4W, (C) L5W, (D) L11W and (E) L12W to LPS. Isothermal calorimetric titration of LPS with peptides in 10 mM sodium phosphate buffer (pH 6.0) at 37 °C. Raw experimental data of LPS titration by peptides and calorimetric titration curve for the binding of peptides to LPS.

Table 2 Thermodynamic parameters of the interactions of peptides with LPS.

I1W I4W L5W L11W L12W

N

Ka (lM1)

DH (kcal mol1)

DS (kcal mol1 deg1)

DG (kcal mol1)

Kd (lM)

0.99 ± 0.03 0.82 ± 0.01 1.03 ± 0.02 2.51 ± 0.04 2.08 ± 0.10

3.34 ± 2.74 2.64 ± 1.16 1.43 ± 3.32 0.21 ± 0.5 0.60 ± 2.24

10.29 ± 0.69 19.72 ± 0.51 8.72 ± 0.03 5.60 ± 0.01 8.01 ± 0.07

3.21 3.53 1.30 6.32 3.82

108.48 110.15 56.82 239.44 149.44

0.29 0.37 0.69 4.76 1.67

1657 cm1 in L5W, 1656 cm1 in L11W and 1659 cm1 in L12W, which is typical for a-helical structures. In addition, a small fraction of unordered conformation is characterized by a band at approximately 1640 cm1. These spectra can be assigned to 50– 80% a-helical structures, which are consistent with their CD spectrum data. 3.5. Disaggregation of LPS aggregates by peptides High binding affinity to and insertion into the hydrophobic lipid A domain of LPS by the peptides may cause structural changes in LPS. We examined the perturbation of LPS using DLS and the fluorescence of FITC-conjugated LPS (FITC-LPS). FITC fluorescence intensity is largely quenched in FITC-LPS solution due to the self-

association of LPS molecules. The addition of proteins or peptides causing dissociation of LPS-aggregated structures may enhance fluorescence intensity [31]. Fig. 7A shows that a significant change occurred in the fluorescence intensity of FITC-LPS with increasing concentrations of I1W and I4W in a dose-dependent manner but that L5W, L11W and L12W only caused a slight increase in the fluorescence intensity of FITC-LPS, indicating that the interaction of I1W and I4W with LPS resulted in the dissociation of LPS aggregates. To further validate the ability of the peptides to dissociate LPS aggregates, dynamic light scattering (DLS) measurements were performed. As shown in Fig. 7B, LPS was initially present in two sizes with average diameters of 100 and 7806 nm. The addition of the I1W and I4W peptides led to the dissociation of the larger



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Fig. 6. Circular dichroism spectra of peptides in 10 mM PBS (A) and with 0.22 mM LPS micelles (B). Peptides exhibit a random structure in water, whereas they adopt a-helical structures in the presence of LPS micelles. (C) Fourier transform infrared spectra in the range of the amide I band. The positions of the peak maxima can be assigned to different secondary structures due to different LPS binding: a-helix between 1651 and 1659 cm1, unordered conformation at 1640 cm1.

Fig. 7. Dissociation of LPS micelles by peptides. (A) Changes in the fluorescence intensity of FITC–conjugated LPS in the presence of peptides; data are expressed as the mean ± SE of 6 independent tests. Size distribution of LPS micelles in the absence (B) and presence of the peptides I1W (C), I4W (D), L5W (E), L11W (F) and L12W (G).


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aggregates of LPS into smaller-sized aggregates, with 680 nm aggregates constituting the most abundant particles (Fig. 7C and D). However, in the presence of L5W, L11W and L12W, the change in the size of the LPS aggregates was smaller (Fig. 7E–G). 3.6. Peptides inhibit cytokine secretion induced by LPS To assess the potential cytotoxicity of the peptides against macrophage, RAW264.7 cells were treated with different concentrations of peptides (0–160 lM) for 1, 6 or 24 h. RAW264.7 cell viability was subsequently evaluated using the MTT method, which is based on the reduction of 3-(4,5-dimethyl-2-thizolyl)- 2,5diphenyl-2H-tetrazolium bromide into formazan dye by the mitochondria of living cells. The MTT assay data indicated that the five peptides rapidly reduced the viability of RAW264.7 cells in a dosedependent but non-time-dependent manner (Fig.8A–C). When the cells were treated with the peptide for 1 h, the peptide showed potent cytotoxicity comparable with other incubating periods, as indicated by the similar curve shape. Within 24 h, at concentrations of 5 lM for I1W, I4W, and L5W and at concentrations of 20 lM for L11W and L12W, the viabilities were largely unaffected (higher than 70% of the total number of cells). To examine whether the tested peptides were able to block the inflammatory responses induced by LPS, RAW264.7 cells were treated with LPS (100 lg/ml) and 5 lM I1W, I4W and L5W or 20 lM L11W and L12W (the concentration with cell viability more than 70%). The levels of secreted IL-6 and TNF-a were measured by ELISA. Fig. 8 D–E shows that LPS significantly (p < 0.01) induced TNF-a and IL-6 production by RAW264.7 cells; however, all peptides at the concentration with cell viability of 70% significantly (p < 0.01) blocked the TNF-a and IL-6 production elicited by LPS.

I1W inhibited the production of LPS-induced TNF-a and IL-6 by up to 80% and 69% of the levels measured without peptide, respectively, whereas the respective inhibition ratios were 41% and 51% for L12W and intermediate for the other three peptides. I4W, compared with I1W, showed low inhibition of TNF-a production. The five peptides exhibited lower activities than polymyxin B, the gold standard for cationic LPS-neutralizing peptides. 4. Discussions Tryptophan residues show a strong preference for the interfacial region of biological membranes, and this property endows Trp-containing peptides with the unique ability to interact with the surface of bacterial cell membranes [10,11,32]. In our previous study, we designed a series of active, membrane-associated Trp-rich peptides to reveal a complex relationship between the structural factors that contribute to antimicrobial activity and the features that determine the hemolytic activity associated with Trp residues [16,17]. We found that five Trp-containing antimicrobial peptides, I1W, I4W, L5W, L11W and L12W, exhibit high activity against Gram-positive bacteria, but L5W, L11W and L12W were inactive against Gram-negative bacteria, particularly against E. cloacae, K. pneumonia and P. mirabilis, although they have similar amino acid compositions (I1W and I4W: 6K, 1W, 1S, 3L and 2I; L5W, L11W and L12W: 6K, 1W, 1S, 2L and 3I), the same length, net positive charge and hydrophobicity, and only the site of the Trp residues is different. For most antimicrobial peptides that are not active against Gram-negative bacteria, the outer membrane is believed to be a major barrier [18,22,33]. The compound 1-N-phenylnapthylamine (NPN), a hydrophobic fluorescent probe, is used to determine changes in the outer membrane permeability of Gram-negative bacteria. Our results showed that I1W and I4W

Fig. 8. (A)–(C): Cytotoxicity of the peptides I1W, I4W, L5W, L11W and L12W in 1 h, 6 h and 24 h. Cells were cultured and treated with various concentrations of peptides (0– 160 lM) for 1 h, 6 h and 24 h, and then subjected to MTT assay. (D) and (E): Production of TNF-a and IL-6. RAW264.7 cells were stimulated with LPS at 100 lg/ml and the peptides I1W (5 lM), I4W (5 lM), L5W (5 lM), L11W (20 lM) and L12W (20 lM) for 24 h. TNF-a and IL-6 was measured by ELISA. The numbers above the bars represent the average inhibition as a result of peptide treatment and the standard errors. **Significantly different from the untreated control (p < 0.01). Each value represents the mean of three independent experiments.



significantly increased the fluorescence intensity of NPN in a concentration-dependent manner in Gram-negative bacteria. In contrast, L5W, L11W and L12W induced a slight increase in the fluorescence intensity of NPN in a concentration-dependent manner in only E. coli cells but were unable induce NPN uptake in the tested Gram-negative bacteria E. cloacae, K. pneumoniae and P. mirabilis. This result suggests that I1W and I4W significantly increase the outer membrane permeability of Gram-negative bacteria but that L5W, L11W and L12W only slightly change the outer membrane permeability of Gram-negative bacteria (E. coli) and even completely prevent extracellular molecules, including AMPs, from entering into E. cloacae, K. pneumoniae and P. mirabilis cells. This is demonstrated by the finding that L5W, L11W and L12W only slightly disrupt the LPS model membrane that mimics the outer membrane of Gram-negative bacteria (calcein release was less than 40%) and that I1W and I4W cause evident calcein leakage from the LPS model membrane, with a calcein release of 100%. Furthermore, in contrast with intact bacteria, L5W, L11W and L12W display a significant bactericidal activity on Gram-negative bacteria spheroplasts that lack the cell wall, particularly E. cloacae, K. pneumoniae and P. mirabilis spheroplasts. The MIC value decreased by 2–8-fold under these conditions. I1W and I4W exhibited similar bactericidal activity on the intact bacterial cells and the spheroplasts of Gram-negative bacteria. Our data suggest that the bactericidal activity of the Trp-containing peptides is related to their ability to traverse the LPS outer membrane. The outer membrane of Gram-negative bacteria is a major barrier that prevents the Trp-containing peptides L5W, L11W and L12W from reaching the inner bacterial membrane. The membrane disruption was closely related to the antimicrobial activity. A direct correlation was found between the bactericidal activity of the peptides and their ability to disrupt the inner membrane of Gram-negative bacteria. All five of the Trp-containing peptides significantly induced the depolarization of the cytoplasmic membrane of E. coli spheroplasts and increased cytoplasmic membrane permeability at a concentration of 1 MIC. This disruption of the cytoplasmic membrane was also confirmed using an indirect measure, the ability of the peptides to induce the release of the selfquenching fluorescent dye calcein from a bacterial mimic membrane. All five peptides caused the total release of calcein from negatively charged LUVs composed of PG/PC, reflecting the membrane perturbation and selectivity of the peptide–membrane interaction with the model membrane [34]. This evidence strongly suggests that the major target of the Trp-containing peptides is the inner membrane of Gram-negative bacteria. The low bactericidal activity of L5W, L11W and L12W against Gram-negative bacteria is the result of their inability to traverse the LPS outer membrane. Once across the outer membrane, the peptides would then have the capability to reach and permeate the inner bacterial membrane to begin the process described as self-promoted uptake [35]. The outer membrane of Gram-negative bacteria consists predominantly of anionic LPS that is composed of a hydrophobic region, termed lipid A, and a saccharide region (O-polysaccharide chain and core region) [36]. The anionic and amphiphilic nature of lipid A enables it to interact with cationic and amphipathic molecules [37]. We have observed a high binding affinity of the five Trp-containing peptides to LPS-outer membrane of Gram-negative bacteria. The zeta potential suggests that electrostatic interactions initially play an important role in peptide binding. The strong cationicity (net positive charge of 6) of the five peptides neutralizes negative charges, resulting in zeta potential values dramatically increased toward positive values. But the charge overcompensation occurs for E. coli cells and five peptides at concentrations of 150 lM. Thus, the five Trp-containing peptides not only bind to the surface of the outer membrane of E. coli, neutralizing all charges, but also insert into the outer membrane of E. coli, due to

11

hydrophobic interactions [38,39]. In addition, charge overcompensation occurs for I1W at concentrations of 300 lM with the other three Gram-negative bacteria, E. cloacae, K. pneumoniae and P. mirabilis, and for I4W at concentrations of 300 lM with K. pneumoniae and P. mirabilis. However, L5W, L11W and L12W exhibit an incomplete neutralization of negative charges in the outer membrane of E. cloacae, K. pneumoniae and P. mirabilis, suggesting that these peptides might not fully access the negative charges of the LPS aggregates. This explanation may be one of the reasons for the lower antimicrobial activity of these peptides against E. cloacae, K. pneumoniae and P. mirabilis compared with the antimicrobial activity against E. coli. In accordance with the zeta-potential results, an exothermic process with negative enthalpy changes was observed in all tested peptides after each titration step by ITC and binding saturations were observed at the highest molar ratio for L5W, L11W and L12W (peptide:LPS = 3.5) and the lowest molar ratio for I1W and I4W (peptide:LPS = 1.5). This also indicates extensive electrostatic-driven peptide binding to LPS. In the overall titration process of I1W and I4W, the LPS-peptide interactions underwent an endothermic reaction, demonstrating the penetration of the peptides into the hydrophobic core of the LPS, except for the electrostatic interactions between the positive charges of the peptide and the negative charges of LPS. I1W and I4W interacted with LPS with submicromolar affinity with a low Kd value, whereas L11W and L12W exhibited a comparatively much weaker binding to LPS with a 5–15-fold Kd. Remarkably, the active peptides I1W and I4W, as well as the inactive L5W, L11W and L12W, assume helical conformations in LPS micelles, although the a-helical contents are different. In other words, irrespective of their activity, the interactions of these peptides with LPS induce conformational transitions into helical states. However, it is not clear from the CD or IR studies whether the site of the Trp residue is responsible for the a-helical content of these peptides in LPS micelles. The direct binding of the peptides to LPS might be important for the bactericidal activity of cationic peptides in Gram-negative strains [18,40]. However, following the disorganization of the LPS leaflet, a concomitant disturbance at the outer membrane level will be helpful to facilitate the entrance of the peptide [41]. Consistent with this finding, we found that the activity of these Trpcontaining peptides on Gram-negative bacteria depends on the outer membrane destabilization process. The addition of peptides largely enhanced the fluorescence intensity of the FITC-LPS solution, suggesting that I1W and I4W cause the dissociation of LPS aggregated structures. Moreover, dynamic light scattering measurements also showed that I1W and I4W lead to the dissociation of larger aggregates of LPS into smaller-sized aggregates, with 680 nm aggregates constituting the most abundant particles. However, in the presence of L5W, L11W and L12W, the change in the size of LPS aggregates was smaller. Our data indicate that the potential mechanisms of action of I1W and I4W at the molecular level involve a ‘‘self-promoted uptake” pathway [35]: the first step is the electrostatic approach of the peptide toward LPS; the peptide then dissociates the LPS aggregates, resulting in the disorganization of the LPS leaflet and promoting the passage of the peptide across the outer membrane into the bacterial phosphatidylglycerol-rich membrane leaflets, disturbing the cytoplasmic membrane. However, despite a higher affinity for LPS, L5W, L11W and L12W cannot destabilize LPS aggregates as demonstrated by DLS measurements and the fluorescence intensity of FITC-LPS. LPS aggregates limit the ability of the peptides to insert into the cytoplasmic membrane, which serves as their major target. This mechanism might be important for the bactericidal activity of the Trp-containing peptides in Gramnegative bacteria. It may be noted that AMPs may neutralize the toxicity of LPS through their interactions with the outer membrane of


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Gram-negative bacteria, further inhibiting the activation of a cell surface receptor, TLR4, thus inhibiting the production of inflammatory cytokines [42–44]. Five Trp-containing peptides can bind to LPS by electrostatic interaction, inhibiting the production of the proinflammatory cytokines TNF-a and IL-6 in LPS-stimulated primary rat macrophages RAW264.7. Andrä et al. reported on LPS interactions with the peptide NK-2, demonstrating both hydrophobic and electrostatic interactions to be necessary for efficient LPS neutralization [45]. Although L5W, L11W and L12W have no bactericidal activities against the Gram-negative bacteria E.cloacae, K. pneumoniae and P. mirabilis due to their low ability to destabilize the LPS outer membrane, their positive charges (+6) and high hydrophobicity allow them to perform efficient neutralization of LPS. In addition, a previous investigation indicated that conformational transition in peptide/LPS complexes was correlated with anti-inflammatory effects [46]. In this context, it is worth noting that the LPS-induced conformational transition of five Trpcontaining peptides may also contribute to peptide antiendotoxic effects. LPS-stimulated proinflammatory responses by macrophages depend on the physical state of LPS, the aggregated state of LPS helps serum proteins to recognize and transfer it to cell membrane receptors [47,48]. I1W inhibits the LPS-induced inflammatory response at a high level, which is also responsible for its ability to dissociate the aggregated state of LPS. Our data suggest that Trp-containing antimicrobial peptides are promising therapeutic candidates for the treatment of infections by Gramnegative bacteria (I1W and I4W) and of LPS-induced inflammatory response (I1W, I4W, L5W, L11W and L12W).

5. Conclusion In this study, we have built upon a series of synthesized Trpcontaining peptides, which previously showed significantly different antimicrobial activity against Gram-negative bacteria despite their similar components and amino acid sequences and identical net positive charge and hydrophobicity, to investigate the effect of the locations of the Trp residues on the antibacterial property of AMPs. Here, we demonstrate that the Trp-containing peptides show stronger interaction with the LPS outer membrane of the Gram-negative bacteria and higher bactericidal activities when Trp residues are located in the amino terminal (I1W and I4W) as opposed to the carboxyl terminal (L11W and L12W). I1W and I4W kill Gram-negative bacteria by a ‘‘self-promoted uptake” pathway in which the peptides first approach LPS by electrostatic forces and then dissociate the LPS. This process results in disorganization of the LPS leaflet and facilitates the ability of the peptide to cross the outer membrane into the inner membrane and disrupt the cytoplasmic membrane. Although L5W, L11W and L12W bind strongly to LPS bilayers and depolarize bacterial cytoplasmic membranes, similar to I1W and I4W, they are unable to destabilize LPS aggregates and traverse through the tightly packed LPS molecules. Five Trp-containing peptides can inhibit the LPS-induced inflammatory response because of their strong binding to LPS. These results suggest that Trp-containing peptides I1W and I4W are promising therapeutic candidates not only for use in traditional therapeutic approaches, such as infections in hospitals and other healthcare facilities associated with Gram-negative bacteria, but also for the treatment of septic shock syndrome caused by LPS.

Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 31272314) and the Program for Liaoning Innovative Research Team in University (LT2015015).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2016.01. 019.

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Comparative Evaluation of the Antimicrobial Activity of Different Antimicrobial Peptides against a Range of Pathogenic Bacteria.

Effects and mechanisms of the secondary structure on the antimicrobial activity and specificity of antimicrobial peptides.

Canine antimicrobial peptides are effective against resistant bacteria and yeasts.

Proline-rich antimicrobial peptides: potential therapeutics against antibiotic-resistant bacteria.

Antimicrobial Activity of Cationic Antimicrobial Peptides against Gram-Positives: Current Progress Made in Understanding the Mode of Action and the Response of Bacteria.

Antimicrobial peptides expressed in medicinal maggots of the blow fly Lucilia sericata show combinatorial activity against bacteria.

An in Vitro Experimental Study on the Antimicrobial Activity of Silicone Oil against Anaerobic Bacteria.

Antimicrobial activity, improved cell selectivity and mode of action of short PMAP-36-derived peptides against bacteria and Candida.

Antimicrobial activity against periodontopathogenic bacteria, antioxidant and cytotoxic effects of various extracts from endemic Thermopsis turcica.

Antimicrobial activity of Antrodia camphorata extracts against oral bacteria.

Protective effects of human milk antimicrobial peptides against bacterial infection.

Activity of the investigational fluoroquinolone finafloxacin and seven other antimicrobial agents against 114 obligately anaerobic bacteria.

Antimicrobial activity of tea catechin against canine oral bacteria and the functional mechanisms.

Cathelicidin Antimicrobial Peptides with Reduced Activation of Toll-Like Receptor Signaling Have Potent Bactericidal Activity against Colistin-Resistant Bacteria.

Nano-engineering the Antimicrobial Spectrum of Lantibiotics: Activity of Nisin against Gram Negative Bacteria.

Antimicrobial Peptides Targeting Gram-Positive Bacteria.

Antimicrobial activity of the essential oil of Tetradenia riparia (Hochst.) Codd. (Lamiaceae) against cariogenic bacteria.

Antimicrobial Activity of the Essential Oil of Plectranthus neochilus against Cariogenic Bacteria.

The in Vitro Antimicrobial Efficacy of PDT against Periodontopathogenic Bacteria.

Does human saliva decrease the antimicrobial activity of chlorhexidine against oral bacteria?

β-Boomerang Antimicrobial and Antiendotoxic Peptides: Lipidation and Disulfide Bond Effects on Activity and Structure.

Impact of interspecific interactions on antimicrobial activity among soil bacteria.

Antimicrobial Activity of Novel Synthetic Peptides Derived from Indolicidin and Ranalexin against Streptococcus pneumoniae.

Inhibitory Effects of Antimicrobial Peptides on Lipopolysaccharide-Induced Inflammation.