A helix-PXXP-helix peptide with antibacterial activity without cytotoxicity against MDRPA-infected mice

Biomaterials 35 (2014) 1025e1039

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A helix-PXXP-helix peptide with antibacterial activity without cytotoxicity against MDRPA-infected mice Jong-Kook Lee a, Seong-Cheol Park a, Kyung-Soo Hahm a, b,1, Yoonkyung Park a, c, * a

Research Center for Proteinaceous Materials (RCPM), Chosun University, Kwangju 501-759, Republic of Korea Bioleaders Corporation, 559 Yongsan-Dong, Yusong-Ku, Daejeon 305-500, Republic of Korea c Department of Biotechnology & BK21-Plus Research Team for Bioactive Control Technology, Chosun University, Gwangju, Republic of Korea b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 September 2013 Accepted 8 October 2013 Available online 28 October 2013

In response to the growing problem of multidrug-resistant pathogenic microbes, much attention is being paid to naturally occurring and synthetic antimicrobial peptides (AMPs) and the effects of their structural modification. Among these modifications, amino acid substitution is a simple approach to enhancing biological activity and reducing cytotoxicity. An earlier study indicated that HPA3, an analog of HP (2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1, forms large pores and shows considerable cytotoxicity. However, HPA3P, in which a proline (Pro) is substituted for glutamic acid (Glu) at position 9 of HPA3, shows markedly less cytotoxicity. This may be attributable to the presence of a Prokink into middle of the HPA3P structure within the membrane environment. Unfortunately, HPA3P is not an effective antibacterial agent in vivo. We therefore designed a helix-PXXP-helix structure (HPA3P2), in which Pro was substituted for the Glu and phenylalanine (Phe) at positions 9 and 12 of HPA3, yielding a molecule with a flexible central hinge. As compared to HPA3P, HPA3P3 exhibited dramatically increased antibacterial activity in vivo. ICR mice infected with clinically isolated multidrug-resistant Pseudomonas aeruginosa showed 100% survival when administered one 0.5-mg/kg dose of HPA3P2 or three 0.1-mg/kg doses of HPA3P2. Moreover, in a mouse model of septic shock induced by P. aeruginosa LPS, HPA3P2 reduced production of pro-inflammatory mediators and correspondingly reduced lung (alveolar) and liver tissue damage. The changes in HPA3 behavior with the introduction of Pro likely reflects alterations of the mechanism of action: i) HPA3 forms pores in the bacterial cell membranes, ii) HPA3P permeates the cell membranes and binds to intracellular RNA and DNA, and iii) HPA3P2 acts on the outer cellular membrane component LPS. Collectively, these results suggest HPA3P2 has the potential to be an effective antibiotic for use against multidrug-resistant bacterial strains. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Helix-PXXP-helix motif HPA3P2 Multidrug-resistant clinical isolated P. aeruginosa Antimicrobial peptide Septic shock mice Antibiotic drug

1. Introduction The frequent use of antibiotics for biomedical and agricultural purposes has led to the emergence of numerous antibiotic-resistant pathogen strains. As a result, infections caused by multidrugresistant bacteria have become a significant problem, especially in hospitals [1,2]. For example, multidrug-resistant Pneumococcus (similar to Pseudomonas aeruginosa) can cause asthma, cystic fibrosis (CF), emphysema and chronic obstructive pulmonary disease (COPD) [3e5]. Consequently, development of a new generation of antimicrobial agents to replace those no longer effective is

* Corresponding author. Research Center for Proteinaceous Materials (RCPM), Chosun University, Kwangju 501-759, Republic of Korea. Tel.: þ82 622306854; fax: þ 82 622256758. E-mail address: [email protected] (Y. Park). 1 Present address: Bioleaders Corporation, Republic of Korea. 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.10.035

urgently needed. Among the promising candidates are antimicrobial peptides (AMPs), which are host defense molecules found in a wide variety of invertebrate, plant and animal species [6,7]. AMPs typically show potent antimicrobial activity against a broad spectrum of microorganisms [8]. The characteristics they generally share include amphipathicity, short length (w60 amino acids), net cationic charge and rapid killing kinetics [9]. Although their precise mechanisms of action are not fully understood, their main cellular targets are known to be the plasma membrane and cytosolic components [10]. To reach both targets, cationic AMPs are electrostatically attracted to the negatively charged lipopolysaccharide (LPS)-containing outer membrane in Gram-negative bacteria [11] and lipoteichoic acid-containing peptidoglycan [12] in Gram-positive bacteria. LPS is composed of a core oligosaccharide, lipid A, and an O-specific chain Ref. [13] and is essential for bacterial growth. Importantly, macrophages stimulated by LPS during bacterial infection release pro-inflammatory cytokines such

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as TNF-a, IL-1 and IL-6 into the blood, which can result in septic shock [14,15]. This makes LPS an excellent target for AMPs, which would have the potential to both directly inhibit bacterial growth and neutralize released LPS. On the other hand, Gram-positive bacteria are covered by peptidoglycan, which is composed of sugar and amino acid polymers, outside of the plasma membrane [16]. Use of antibiotics that act by inhibiting peptidoglycan production (e.g., penicillin) has led these bacteria to develop resistance through, for example, expression of penicillin-binding proteins or transpeptidases [17,18]. For that reason, the activity of AMPs at the bacterial cell wall must be better understood before effective antibiotics can be developed. We previously reported isolating the AMP HP (2-20) from the Nterminal region of the Helicobacter pylori ribosomal protein L1 [19]. This peptide exhibits antimicrobial activity against Gram-negative bacteria, Gram-positive bacteria and fungi without cytotoxicity against mammalian cells. However, when HP (2-20) was modified to enhance its antimicrobial activity, the modified peptide following substitution of tryptophan (Trp) for glutamine (Gln) and aspartic acid (Asp) at positions 16 and 18, respectively, was found to be hemolytic and cytotoxic in mammalian cells [20]. HPA3 peptide was then designed to reduce cytotoxicity in hRBCs and mammalian cells, while maintaining or increasing its antimicrobial activity and improving its structural flexibility. Previous studies have shown that proline (Pro) induces a folded conformation and provides flexibility to peptides and proteins. Accordingly, Pro has been used in the design of a number of AMP analogs [21,22]. In HPA3, we substituted Pro for glutamic acid (Glu) at position 9 (HPA3P) or Glu and phenylalanine (Phe) at positions 9 and 12 (HPA3P2). The antibiotic and cytotoxic activities of the peptides were then measured against Gram-positive and Gram-negative multidrugresistant bacteria, in vitro and in vivo, and their effects on production of inflammation-related proteins and septic shock were also tested. Finally, the mechanisms of action of the peptides were investigated.

2. Materials and methods 2.1. Materials L-a-Phosphatidylethanolamine (PE, from Escherichia coli), sphingomyelin (SM, from porcine brain), cholesterol (CH, from ovine wool), L-a-phosphatidylglycerol (PG, from E. coli) and L-a-phosphatidylcholine (PC, from chicken egg) were obtained from Avanti Polar Lipids (Alabaster, AL). 3,30 -diethylthio-dicarbocyanineiodide (DiSC3-5), carboxytetramethyl rhodamine succinimidylester (TAMRA) and calcein were from Molecular Probes (Eugene, OR). Plasmid DNA (pTYB2) was purchased from New England Biolabs Ltd. Ergosterol, E. coli O111:B4 lipopolysaccharide (LPS), erythromycin, ampicillin, ciprofloxacin, rifampin and vancomycin were from SigmaeAldrich Co. All other reagents were of analytical grade.

2.3. Peptide synthesis and purification Peptides were synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) solidphase methods on Rink amide 4-methyl benzhydrylamine resin (Novabiochem) (0.55 mmol/g) with a Liberty microwave peptide synthesizer (CEM Co. Matthews, NC). To generate N-terminal fluorescently-labeled peptides, resin-bound peptides were treated with 20% (v/v) piperidine in dimethylformamide (DMF) to remove the Fmoc protection groups from the N-terminal amino acids. Resin-bound peptides were then reacted with rhodamine-SE in DMF (3e4 eq.) containing 5% (v/v) diisopropylethylamine. After gently mixing for 24 h in the dark, the resins were washed thoroughly, first with DMF and then with dichloromethane (DCM). Thereafter, the peptides were cleaved from their respective resins, precipitated with ether and extracted [23]. The crude peptides were purified using reversed-phase preparative HPLC on a Jupiter C18 column (250  21.2 mm, 15 mm, 300 Å) with an appropriate 0e 60% acetonitrile gradient in water containing 0.05% trichloroacetic acid. Peptide purity was then determined by analytical reversed-phase HPLC on a Jupiter proteo C18 column (250  4.6 mm, 90 Å, 4 mm). The molecular masses of the peptides were confirmed using matrix-assisted laser desorption ionization mass spectrometry (MALDI Ⅱ, Kratos Analytical Ins.). 2.4. Microorganisms E. coli (ATCC 25922), Staphylococcus aureus (ATCC 25923) and P. aeruginosa (ATCC 15692) were obtained from the American Type Culture Collection. Bacillus subtilis (KCTC 1998) was from the Korean Collection for Type Cultures. E. coli CCARM 1229, E. coli CCARM 1238, S. aureus CCARM 3089, S. aureus CCARM 3114, Salmonella typhimurium CCARM 8009 and S. typhimurium CCARM 8013 were distributed from the Culture Collection of Antibiotic Resistant Microbes at Seoul Women’s University, Korea. P. aeruginosa 4007, 3547 and 4891 are resistant strains isolated from patients with otitis media in a hospital. 2.5. Microdilution assay The antimicrobial activities of HPA3, HPA3P and HPA3P2 were determined in microdilution assays. Briefly, two-fold serial dilutions of each peptide and antibiotic (ampicillin, erythromycin and piperacillin) were added in duplicate to media containing inoculants of the test bacteria (5  105 cfu/ml) at mid-logarithmic growth phase. The samples were then incubated for 18e24 h at 37  C, after which the minimum inhibitory concentrations (MICs) of the peptides and antibiotics were determined by measuring the absorbance level at 650 nm. The lowest concentration of peptide or antibiotic that completely inhibited growth is defined as the MIC [20]. 2.6. Hemolysis Fresh human red blood cells (hRBCs) from healthy donors were centrifuged at 800  g and then washed with PBS until the supernatant was clear. Two-fold serial dilutions of each peptide in PBS were added to the wells of a 96-well plate before addition of hRBCs to a final concentration of 8% (v/v). The samples were then incubated with mild agitation for 1 h at 37  C and centrifuged at 800  g for 10 min, after which the absorbance of the supernatant was measured at 414 nm. All measurements were made in triplicate [24], and the percentage of hemolysis was calculated using the following equation: % hemolysis ¼ ½ðAbs414 in the peptide solution  Abs414 in PBSÞ=  ðAbs414 in 0:1 % Triton  X100  Abs414 in PBSÞ  100 where 100% hemolysis is defined as the absorbance measured from hRBCs exposed to 0.1% Triton X-100 and zero hemolysis is characterized as hRBCs alone in PBS. 2.7. Cell culture and cytotoxicity

2.2. Ethics statement This study was approved by the institutional ethics committee, and all healthy donors provided written informed consent before treatment. We processed to the Ethical standards of the Institutional Ethics Committee of Chosun University and to the checklist for ethical consideration of cytotoxicity studies (https://www.cre.or.kr/ article/policy/1382313). Human red blood cells (RBCs) were obtained from blood freshly collected from healthy donors at the Chosun University Hospital in Kwangju (Republic of Korea). Moreover, this study received ethics approval from the Institutional Ethics Committee of Chosun University. The authors of this article were blinded to all personal data from the donors, and all blood donors remained anonymous. All procedures were carried out according to rules provided by the Institutional Ethics Committee of Chosun University. Samples of blood were obtained from 5 healthy donors. The samples were immediately stored at 4  C until needed. The mouse studies were carried out in accordance with the National Institutes of Health guidelines for the ethical treatment of animals. All animal procedures conformed to guidelines from Center for Experimental Animal of Chosun University. All work was covered by Center for Experimental Animal of Chosun University licences: CUCEA 00001, “Antibacterial activity of AMP (HPA3 series)” with approval by the Chosun University ethical review panel.

To examine the cytotoxic effects of the peptides, HaCaTs (cultured human keratinocytes) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with antibiotics (100 U/ml of penicillin, 100 mg/ml of streptomycin) and 10% fetal calf serum at 37  C in a humidified chamber under a 5% CO2 atmosphere. Growth inhibition was evaluated using MTT assays to assess cell viability. Cells were seeded into a 96-well plate at a density of 4  103 cells/well and incubated for 24 h. Two-fold serial dilutions of peptides in DMEM were then added to the plate, and the cells were incubated for an additional 24 h at 37  C. Thereafter, 10 ml of MTT (5 mg/ ml) were added to each well, and the plate incubated for 4 h. The supernatants were then aspirated, and 50 ml of DMSO were added to each well to dissolve any remaining precipitate, and the absorbance at 570 nm was measured using a microtiter reader [25]. 2.8. Radial diffusion assay Pre-grown bacteria in 3% (w/v) LuriaeBertani (LB, for E. coli and B. subtilis) broth, trypticase soy broth (TSB, S. aureus) or nutrient broth (NB, for P. aeruginosa) containing 0.5% NaCl were washed once with 10 mM sodium phosphate (pH 7.4). Fifteen ml of underlay agarose gel containing 0.03% (w/v) TSB, 1% (w/v) low electroendosmosis type agarose and 5  105 colony forming units (cfu)/ml were poured into a Petri dish and solidified to a gel. After 8 ml of peptide solution were added at

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the indicated concentrations to punched wells with a diameter of 3 mm, the plate was incubated at 37  C for 3 h. The gel was then covered with an overlay gel solution containing 6% culture media and 1% agarose, after which the plate was further incubated at 37  C for 24 h until a clearance zone appeared.

mid-logarithmic growth phase at 37  C were suspended in 10 mM sodium phosphate buffer to an OD600 of 0.15, and 1/2 MIC of each peptide was added. After incubation for 30 min at 37  C, the cells were negatively stained with 1% (w/v) uranyl acetate (UAC) in the absence or presence of each peptide [30] (B).

2.9. Survival rate and histological analysis in an infection disease model

2.14. DNA-binding assay

The survival rate among 6-week-old ICR mice intraperitoneally injected with multidrug-resistant P. aeruginosa 4007 cells (MDRPA; 6  107 cfu/ml) from patients with otitis media (Chunnam National University) was examined. Following intraperitoneal injection of HPA3P (single doses of 0.5 or 1 mg/kg), HPA3P2 (single doses of 0.25 or 0.5 mg/kg; doses of 0.1 mg/kg two or three times per day) or DMEM (negative control), morbidity and mortality were recorded daily over a 10-day period. Histological changes in the lungs of ICR mice treated with HPA3P2 (0.25 or 0.5 mg/kg) following injection with MDRPA were assessed by hematoxylin and eosin (H&E) staining. Briefly, organs were dissected from ICR mice and washed with PBS, after which they were fixed in 4% paraformaldehyde for 24 h at 4  C, dehydrated through a 50e100% ethanol series for 2 h each, and bathed in xylene three times for 20 min each to remove the paraffin. The paraffin-embedded samples were then sectioned at a thickness of 4 mm (Microtome, Thermo-scientific) and stained with H&E. Another group was intraperitoneally injected with D-galactosamine (D-GalN, 500 mg/kg) with or without P. aeruginosa LPS (16.7 mg/kg) to induce acute hepatic damage, and treated with HPA3P2 (0.25 mg/kg) 30 min later. After 2 days, mouse lung and liver tissues were collected, and paraffin sections (4 mm) were stained with H&E and visualized under a fluorescence microscope (Ix71, Olympus, Tokyo, Japan) [26].

Plasmid pTYB2 (NEB Ltd.) was purified using a plasmid extraction kit (ExprepÔ Quick, GeneAll Biotechnology Co., Seoul, Korea), and yeast RNA was isolated using TRI reagent (MRC Inc. Cincinnati, OH). The collected plasmid DNA (200 ng) and yeast RNA (10 mg) were then mixed with increasing amounts of peptide in buffer containing 10 mM Tris (pH 8.0), 1 mM EDTA, 5% glycerol, 20 mM KCl and 50 mg/ml BSA, after which the mixtures were incubated for 10 min at 37  C, subjected to 0.5% or 1% agarose gel electrophoresis in TBE buffer, and stained with ethidium bromide [31]. Gel retardation was visualized under UV illumination using a Bio-Rad Gel Documentation system (UK).

2.10. GFP-expressing E. coli in the abdomen of ICR mice E. coli BL21 (DE3) cells transfected with pET28a-GFP were grown to midlogarithmic phase in LB broth, after which 0.5 mM of IPTG was added to induce expression of GFP. The GFP-labeled E. coli (6  107 cfu/ml) cells were then intraperitoneally injected into ICR mice. One h later, a single dose of HPA3P2 (0.25 or 0.5 mg/kg) was administered. Then after an addition 6 h, the stomach, kidney and liver were collected, sectioned at a thickness of 4 mm and visualized using a fluorescence microscope (Ix71, Olympus, Tokyo, Japan) [27].

2.15. Tryptophan (Trp) fluorescence blue shift The fluorescence emission spectra of the Trp residues within the peptides were monitored in aqueous buffer with and without pDNA purified from E. coli. Mixtures of peptide and pDNA (w/w: 0, 0.5, 1, 1.5, 2 or 3) were added to 1 ml of 10 mM HEPES buffer (pH 7.2), after which Trp fluorescence was measured at 280 nm, and the emission spectrum was measured from 300 to 450 nm in 1 nm increments with 1 s signal averaging. The wavelength with the maximum fluorescence emission was plotted or shifted for the peptide concentration. Trp fluorescence measurements were made on a PerkineElmer LS55 fluorometer. 2.16. Inhibition of recombinant GFP expression in E. coli E. coli BL21 (DE3) cells transfected with pET28a-GFP were grown to midlogarithmic growth phase at 37  C, after which aliquots were suspended to an OD600 of 0.15 in 10 mM sodium phosphate buffer containing 10% LB media. Peptides were then added to the mixtures to their 1/4 MIC, 1/2 MIC or MIC. After incubating the mixtures for 30 min, 0.5 mM IPTG was added to induce GFP expression, and the mixture was incubated for an additional 4 h at 37  C. The cells were then centrifuged, washed three times to remove unreacted peptides, lysed by sonication and centrifuged again. The cytosolic proteins in the resultant supernatant were visualized using fluorescence microscopy or 15% SDS-PAGE [27].

2.11. Western blot analysis 2.17. Neutralization of LPS

Western blot analysis was performed as previously described. Briefly, proteins from mouse organs were separated by 15% SDS-PAGE for 3 h and then transferred to a PVDF membrane (Bio-Rad, USA) for 1 h at 90 V. Each membrane was incubated overnight at 4  C with primary antibody in 5% skim milk. The antibodies used were anti-GAPDH (Santa Cruz Biotechnology), anti-TLR4, anti-IL-6, anti-CXCL5 (ABfrontier), anti-NF-kB mouse (Santa Cruz Biotechnology), anti-TNF-a (ABfrontier) and antiIL-1b (ABfrontier). The membranes were then washed with TBST buffer and incubated with secondary antibodies (goat anti-rabbit IgG (HRP)), after which the blots were developed using a Western blot detection kit (ABfrontier) [24].

Peptide neutralization was measured using chromogenic limulus amoebocyte lysate assays [32]. A constant concentration of LPS (1 ng/ml) was incubated with various concentrations of peptide (0e25 mM) at 37  C in the wells of an apyrogenic sterile microtiter plate. Aliquots (50 ml) of this mixture were then added to equal volumes of limulus amoebocyte lysate reagent and incubated for 10 min at 37  C. A yellow color developed upon addition of 100 ml of chromogenic substrate solution. After stopping the reaction by addition of 25% acetic acid, the absorbance was measured at 405 nm.

2.12. Confocal laser-scanning microscopy

2.18. Effect of AMPs on LPS-dependent induction of TNF-a

To analyze the cellular distribution of peptides, E. coli were incubated in the presence of tetraethyl rhodamine (TAMRA)-labeled peptide and observed using a confocal laser-scanning microscope. The cells were inoculated and adjusted using the antimicrobial assay procedure, after which TAMRA-labeled peptides were added to their respective MICs in 100 ml of the cell suspension. After incubation for 30 min, the cells were pelleted by centrifugation at 4,000 rpm for 5 min and washed three times with ice-cold PBS buffer. Localization of TAMRA-labeled peptides was exam}ttingen, ined using an inverted LSM510 laser-scanning microscope (Carl Zeiss, Go Germany) equipped with a helium/neon laser (543 nm line). The resulting images were recorded digitally in a 512  512 pixel format [28].

To examine TNF-a expression induced by E. coli LPS, Raw 264.7 cells (mouse macrophage line) were cultured in DMEM supplemented with antibiotics (100 U/mM penicillin, 100 mg/ml streptomycin) and 10% fetal calf serum at 37  C in a humidified chamber under a 5% CO2 atmosphere. The cells were then plated in 12-well plates (2  105 cells/well) and cultured for 24 h at 37  C/5% CO2. Expression of TNF-a induced by E. coli LPS (10 ng/ml) during 6 h in supplemented DMEM was evaluated in the presence or absence of each peptide (10 mM). Cells were washed once with PBS, after which total RNA was extracted using TRI reagent, and cDNA products were amplified in the presence of 30 and 50 primers (TNF-a, 50 -GACGTGGAACTGGCAGAAGAG-30 and 50 -TTGGTGGTTTGTGAGTGTGAG-30 ; GAPDH, 50 -CCATCAACGACCCCTTCATTGAC-30 and 50 -GGATGACCTTG-CCCACAGCCTTG-30 ). The cycling protocol for the amplification reaction was as follows: 30 min of reverse transcription at 45  C and 5 min of inactivation of RTase at 94  C, followed by 30 cycles of denaturation at 94  C for 40 s, annealing at 62  C (for TNF-a) or 56  C (for GAPDH) for 40 s, and extension at 72  C for 1 min. After the last cycle, the reaction mixture was incubated at 72  C for 5 min and cooled to 4  C. The amplified products were then separated by 1% agarose gel electrophoresis and visualized. E. coli LPS (10 ng/ml)induced production of pro-inflammatory cytokines over the course of 12 h in supplemented DMEM in the presence or absence of HPA3P or HPA3P2 (0e50 mM) was evaluated by measuring the absorbance at 450 nm on a microplate reader (VERSA max microplate reader). TNF-a levels were determined using a TNF-a ELISA kit (Biotechnology Komabiotech) [32,33].

2.12.1. SYTOX green uptake assay E. coli cells were grown to mid-logarithmic phase at 37  C, washed and suspended (2  107 cells/ml) in 10 mM sodium phosphate buffer (pH 7.2), and incubated with 1 mM SYTOX green for 15 min in the dark [29]. AMPs (HPA3, HPA3P, HPA3P2 or Buforin-2) were then added at the appropriate concentrations (0.5e8 mM), and timedependent increases in fluorescence caused by binding of the cationic dye to intracellular DNA were monitored (excitation wavelength, 485 nm; emission wavelength, 520 nm). 2.13. Transmission electron microscopy (TEM) analysis Plasmid pTYB2 was expressed in E. coli cells grown to mid-logarithmic phase at 37  C. The cells were then suspended in 10 mM sodium phosphate buffer to an OD600 of 0.15, lysed by sonication and centrifuged to collect the pTYB2 DNA, which was then negatively stained with 1% (w/v) uranyl acetate (UAC) in the absence or presence of HPA3P (peptide/DNA: 0.5 or 1, w/w) (A). Morphological alteration of E. coli cells was assessed by comparing cell incubated in the absence or presence of HPA3, HPA3P or HPA3P2. E. coli cells grown to

2.19. Circular dichroism (CD) analysis CD spectra were recorded at 25  C on a Jasco 810 spectropolarimeter (Jasco, MD, USA) equipped with a temperature control unit. A 0.1-cm path-length quartz cell containing 30 mM peptide solution was used along with 50% trifluoroethanol (TFE, v/ v), 300 mM LPS, or 30 mM SDS, w/v. At least five scans were acquired and averaged to

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Table 1 Sequences, molecular masses and antimicrobial activities of HPA3, HPA3P and HPA3P2. Designation

HPA3 HPA3P HPA3P2 a b c d

Molecular massa

Sequence

AKKVFKRLEKLFSKIWNWK-NH2 AKKVFKRLPKLFSKIWNWK-NH2 AKKVFKRLPKLPSKIWNWK-NH2

2448.5 2417.2 2367.0

MIC (mM)b Ec

Pa

Sa

Bs

2 2 2

2 2 2

2 4 8

2 1 1

HC10/Hc50c

EC10/Ec50d

49.2/397.7 >800/>800 >800/>800

3.1/64.4 151.5/381.2 180.5/>400

Molecular masses were measured using MALDI-TOF MS/MS. The minimal inhibitory concentration (MIC) is defined as the concentration that completely inhibits growth of E. coli (Ec), P. aeruginosa (Pa), S. aureus (Sa) or B. subtilis (Bs). HC10 and HC50 are the peptide concentrations that respectively induce 10% and 50% hemolysis among human erythrocytes. EC10 and EC50 are the 10% and 50% effective concentrations, respectively. The substituted prolines (P) are in bold.

Table 2 Antibacterial activities of HPA3, HPA3P and HPA3P2 against drug-resistant strains. Strains

MIC (mM) HPA3 SPM

S. aureus CCARM 3090 S. aureus CCARM 3018 S. aureus CCARM 3114 S. aureus CCARM 3126 E. coli CCARM 1229 E. coli CCARM 1238 P. aeruginosa 1034 P. aeruginosa 3904 P. aeruginosa 4007 P. aeruginosa 4891 a b

1 1 1 1 1 1 1 1 1 1

a

HPA3P b

a

MHB

SPM

16 16 8e16 16 8 8 8 16 16 8

1 1 1 1 0.5-1 0.5e1 1 1 1 1

HPA3P2 b

MHB

SPM

32 32 32 16 16 8 8 16 32 8

1 1 1 1 0.5 0.5 2 2 4 4

a

Ampicillin b

b

Erythromycin b

Piperacillin

MHB

MHB

MHB

MHBb

64 64 64 64 32 32 64 64 >64 >64

e e e e >800 >800

>800 >800 >800 >800 e e

e e e

e e e

e e e e e e >400 >400 400 >400

Microdilution assays were performed in 10 mM sodium phosphate (pH 7.4) supplemented with 10% Mueller-Hinton broth (MHB). This assay was followed by NCCLS methods. P. aeruginosa 4007, 3547 and 4891 are resistant-strains isolated from patients with otitis media in a hospital. () No activity.

Fig. 1. Antibacterial activities of HPA3, HPA3P and HPA3P2 were determined in radial diffusion assays. Pre-cultured bacteria were inoculated into 1% agarose gel with 0.03% culture media. After adding the indicated peptide (8 ml) to 4-mm-diameter wells, the plates were incubated for 3 h, after which they were overlayed with 1% agarose with 6% TSB and incubated for an additional 24 h. Clear zones reflect to the inhibitory effect of each peptide (mean values are presented, n ¼ 3).

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Fig. 2. Evaluation of the antibacterial activities of HPA3P and HPA3P2 against MDRPA in a bacterial infection model. (A) Survival rates among ICR mice treated with the indicated peptides. To assess the antibacterial effects of HPA3P and HPA3P2 in vivo, ICR mice were intraperitoneally infected with MDRPA (6  107 CFU/ml). Beginning 1 h later, single or multiple doses of the peptides were intraperitoneally administered at the indicated concentrations. (B) Histology of P. aeruginosa-infected mouse lungs stained with H&E. The lungs were harvested on day 4 after infection.

improve the signal-to-noise ratio at 250-190 nm. Mean residue ellipticities ([q], degcm2dmol1) were calculated using the following equation [34,35]: ½q¼obs =10lc where obs is the measured signal (ellipticity) in millidegrees, l is the optical pathlength of the cell in cm, and c is the concentration of peptide in mol/L (c is the mean residue molar concentration: c ¼ number of constructed residues of peptide  molar concentration of the peptide (mol/L)).

3. Results 3.1. Design and lytic activities of HPA3P2 To examine the effects of three a-helical structures, straight (HPA3), bent (HPA3P) and hinged (HPA3P2), on antibacterial activity and mechanism of action, HPA3, which forms a straight a-

helix in a membrane environment, was used as a parent model peptide. In an earlier report, we introduced a kink or bend into the middle of HPA3 by substituting the Glu residue at position 9 with Pro, resulting in HPA3P [21]. For the present study, we also designed HPA3P2, which has a helix-PXXP-helix structure. By substituting Glu at position 9 and Phe at position 12 with Pro residues, we introduced a flexible central hinge into the structure. Table 1 summarizes the structural characteristics of the three peptides as well as their antibacterial activities against four pathogenic bacteria. As compared to HPA3P and HPA3, HPA3P2 showed similar or slightly greater antibacterial activity against E. coli, P. aeruginosa and B. subtilis, but was less effective against S. aureus. Table 2 shows their activities against 10 antibiotic-resistant bacteria obtained from clinical isolates. Although commercial antibiotics were not active against these multidrug-resistant strains, our AMPs

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Fig. 3. In vivo effect of HPA3P2 on mice infected with GFP-labeled E. coli. Mice were infected with GFP-labeled E. coli. Then after 1 h, HPA3P2 (0.25 and 0.5 mg/kg) was intraperitoneally administered in a single dose. Sections of stomach (A), kidney (B) and liver (C) were visualized under a fluorescence microscope.

showed potent antibacterial activity, with MICs ranging from 1 to 64 mM. Importantly, HPA3P2 induced little or no hemolysis at concentrations up to 800 mM and markedly less HaCaT cell cytotoxicity (about 48.7- to 58-fold less at the EC50) than the other peptides. In radial diffusion assays, HPA3P2 was more active than the other two peptides (Fig. 1). In these assays, the size of the clear zone increased with increasing doses of HPA3P2 such that it was directly proportional to the logarithm of the HPA3P2 dose. The antibacterial activity and cytotoxicity of HPA3P2 were further compared with those of HPA3 and HPA3P in the following examination of their mechanisms of action. 3.2. In vivo activities of HPA3P and HPA3P2 and HPA3P2-mediated protection against septic shock To evaluate the antibacterial activities of HPA3P and HPA3P2 in vivo, MDRPA was inoculated into ICR mice. The mice were

Fig. 4. Western blot analysis of the effect of HPA3P2 on expression of the indicated signaling molecules in lungs from MDRPA-infected ICR mice. Control (A), P. aeruginosa 4007 (B), HPA3P2 0.25 mg/kg, 1 day (C) or HPA3P2 0.25 mg/kg, 7 days (D).

divided into six groups (8 mice/group) and infected by intraperitoneal injection (6  107 cfu/ml). Thereafter, single doses of HPA3P (0.5 or 1 mg/kg) or HPA3P2 (0.25 or 0.5 mg/kg) were administered intraperitoneally. The results are illustrated in Fig. 2A (left panel). Over the subsequent 3 days, all mice in the groups without peptide treatment died. Although mice treated with HPA3P (0.5 or 1 mg/kg) peptide survived somewhat longer than those in the untreated groups, all these mice also died within 3e4 days. By contrast, mice treated with 0.25 mg/kg HPA3P2 showed 50% survival after 10 days, and all mice treated with 0.5 mg/kg HPA3P2 survived for the 10-day observation period plus an additional 4 weeks (data are not shown). Thus HPA3P2 appears to have substantial antibacterial activity against multidrug-resistant bacteria in vivo. We next examined the efficacy of administering multiple lower doses of HPA3P2 to MDRPA-infected mice (Fig. 2A, right panel). When we administered 0.1 mg/kg HPA3P2 two or three times on day 1, the survival rate among mice receiving two doses was 80% over 10 days, and all of the mice receiving three doses survived. These results demonstrate that the antibacterial efficacy of HPA3P2 was enhanced by administration in multiple doses. Moreover, histopathological examination of samples of lung tissue from untreated mice revealed MDRPA-infected lungs to be swollen and showing infiltration by immune cells as well as alveolar hemorrhaging. By contrast, lungs from mice treated with 0.25 or 0.5 mg/ kg HPA3P2 showed dose-dependent improvement (Fig. 2B). To further evaluate the in vivo killing effect of HPA3P2, ICR mice were infected with GFP-labeled E. coli (6  107 cfu/ml), after which a single dose of HPA3P2 (0.25 or 0.5 mg/kg) was administered intraperitoneally. Then after 6 h the mice were sacrificed, and their stomach, kidney and liver were dissected, sectioned and visualized under a fluorescence microscope. As shown in Fig. 3, organs from mice treated with HPA3P2 showed markedly less green fluorescence than those from untreated mice, which strongly fluoresced. The Western blot analysis in Fig. 4 shows that the chemokine CXCL5; the pro-inflammatory cytokines IL-6, IL-1b and TNF-a; NFkB signaling protein; and toll-like receptor-4 (TLR-4) were all significantly induced in mice infected with MDRPA, and that levels

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Fig. 5. Histology of tissues from ICR mice. Histology of liver (A) and lungs (B) from ICR mice intraperitoneally administered PBS (a), 500 mg/kg D-GaIN (b), D-GaIN þ 16.7 mg/kg P. aeruginosa LPS (c) or D-GalN þ P. aeruginosa LPS þ 0.25 mg/kg HPA3P2 (d). On day 2 after treatment, lungs and livers were harvested, sectioned and stained with H&E.

of all these molecules were lower in mice treated with HPA3P2 (0.25 mg/kg) (days 1 and 7 after treatment). This suggests that not only does HPA3P2 possess antibacterial activity against MDRPA, it also has anti-endotoxin activity against LPS. HPA3P2 may thus have the ability to prevent septic shock.

To evaluate the ability of HPA3P2 to prevent septic shock, mice were sensitized with D-GalN and then administered P. aeruginosa LPS to induce septic shock. Histopathological analysis of lung and liver sections taken from control animals challenged with D-GalN/ LPS in the absence of peptide showed the characteristic features of shock, including widespread destruction of the organ architecture and erythrocyte agglutination (Fig. 5). By contrast, tissues from mice treated with HPA3P2 (0.25 mg/kg) were undamaged and appeared similar to those from mice treated with D-GalN only. Based on these results, we suggest that HPA3P2 has potent antiendotoxin activity and could protect against septic shock. 3.3. Mechanisms of action of AMPs

Fig. 6. Localization of rhodamine-conjugated peptides in E. coli cells. E. coli cells were incubated for 10e15 min with the rhodamine-labeled peptides at their MIC and then observed under a confocal laser-scanning microscope.

To gain insight into the mechanisms of action of HPA3, HPA3P and HPA3P2 and better understand the differences in their in vivo effects, we initially used confocal laser-scanning microscopy to compare their distributions within bacterial cells. Fig. 6 shows the localization of the rhodamine-labeled peptides within E. coli cells. Whereas HPA3 was present on the cell surface, HPA3P accumulated in the cytoplasm. HPA3P2 peptide also localized on the surface of bacterial cells. SYTOX green is a fluorescent indicator that produces a strong fluorescent signal upon entering cells and binding to nucleic acids; especially strong signals are produced when the inner membrane of Gram-negative bacteria is permeabilized or disrupted. HPA3 induced a dose-dependent increase in fluorescence intensity (Fig. 7A). Although maximum uptake of HPA3P was observed at 2 mM, the intensity of the induced fluorescence was substantially less than that induced by HPA3 (Fig. 7B). The fluorescence pattern HPA3P is similar to that of buforin-2, an AMP known to permeabilize bacterial cell membranes (Fig. 7D) [36]. Interestingly, HPA3P2 did not induce uptake of SYTOX green, indicating that it did not enter the inner membrane of E. coli (Fig. 7C). These data suggest that HPA3 acts by forming pores in bacterial cell membranes, whereas HPA3P permeates the cell membranes and binds to nucleic acids, and HPA3P2 disrupts the outer membrane.

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Fig. 7. Time-dependent influx of SYTOX green into E. coli cells. E. coli (2  107 cells/ml) were incubated with 1 mM SYTOX green until the basal fluorescence reached a steady state (w15 min). HPA3 (A), HPA3P (B), HPA3P2 (C) or buforin-2 (D) was then added, and the fluorescence was measured at the indicated times (Ex. 485 nm and Em. 520 nm).

Morphological changes in E. coli cells induced by HPA3, HPA3P and HPA3P2 were examined using TEM. After exposure to HPA3 at its 1/2 MIC for 30 min, E. coli exhibited severe surface damage and distorted morphology (compare Fig. 8A and B); cells exposed to HPA3P appeared swollen, without membrane damage (Fig. 8C); and cells exposed to HPA3P2 exhibited no change cell shape, but floating particles were seen in the peripheral regions of the cells (Fig. 8D). Taken together, these results indicate that each of the three peptides exerts its bactericidal effect via a different mechanism. To confirm the different mechanisms of action of HPA3P and HPA3P2, we first used gel retardation assays to evaluate the binding affinity of HPA3P for plasmid DNA (Fig. 9A). When DNA and HPA3P were mixed, the maximum band shift occurred at a mass ratio of 1, indicating formation of a peptide-pDNA complex. On the other hand, buforin-2, used as a control, induced a slower shift, and complete binding was achieved at a mass ratio of 3. The pDNAbinding affinity of HPA3P was also assessed using Trp blue shift assays, since the emission spectrum of the Trp residues in HPA3 peptide shifts toward the blue upon DNA binding (Fig. 9B). The emission spectrum shifted slightly toward blue wavelengths at a mass ratio of 0.5 and a complete shift was seen at a 1:1 (w/w) ratio. The degree to which the emission spectrum shifted with the ratios was in the order, peptide/pDNA: 0.5 > 3>2 > 1.5 > 1, w/w (Fig. 9B). Finally, to directly visualize HPA3P binding to pTYB2, the peptide and the plasmid were mixed together and then examined using TEM. Fig. 9C shows that pTYB2 DNA did not aggregate in the absence of peptide (Fig. 9C, panel 1), but it clumped together when the peptide/DNA ratio was 0.5 or 1, confirming the interaction between HPA3P and pTYB2 DNA (Fig. 9C, panels 2 and 3).

To investigate the effect of intracellular HPA3P on GFP expression in E. coli cells, expressed GFP was visualized using fluorescent microscopy and 15% SDS-PAGE. Fig. 10A shows that untreated cells strongly fluoresced, but that the fluorescence declined as cells were exposed to increasing concentrations of HPA3P. When we used SDS-PAGE to assess GFP expression in E. coli in the presence and absence of the peptide, we found that GFP expression gradually declined with increasing concentrations of HPA3P until, at 1/2 MIC, the GFP band was virtually gone (Fig. 10B). This suggests HPA3P enters E. coli cells, binds to nucleic acids and inhibits protein synthesis. 3.4. LPS-neutralizing activity The LPS-neutralizing activities of HPA3, HPA3P and HPA3P2 were evaluated using LAL assays. All of the peptides strongly interacted with E. coli LPS at lower concentrations than did melittin (positive control) [37], with HPA3P2 showing the greatest affinity (Fig. 11A). Because TNF-a is one of the first pro-inflammatory cytokines secreted by LPS-stimulated immune cells, we also examined the effects of the peptides on the transcription and secretion of TNF-a from RAW 264.7 cells stimulated with 10 ng/ml LPS. We found that after exposing the cells to LPS for 6 h, transcription of TNF-a gene was significantly lower in cells treated with LPS þ peptide than in control cells treated with LPS alone. When applied in the absence of LPS, none of the three peptides stimulated TNF-a expression, indicating that none of the peptides are immunogenic (Fig. 11B). In addition, HPA3P and HPA3P2 both dosedependently inhibited release of TNF-a from RAW 264.7 cells (Fig. 11C). Overall, HPA3P2 showed the strongest binding affinity for

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Fig. 8. Morphological changes in E. coli cells incubated in the absence (A) or presence of HPA3 (B), HPA3P (C) or HPA3P2 (D). After incubation for 30 min, cells were stained with 2% UAC and visualized using TEM. bar ¼ 500 nm.

LPS and the greatest inhibition of TNF-a expression in Raw 264.7 cells. 3.5. Differences in secondary structure in artificial membranemimetic environments Finally, we used CD spectroscopy to examine the secondary structures of HPA3, HPA3P and HPA3P2 in 10 mM sodium phosphate buffer at pH 7.2 (aqueous solution), 50% trifluoroacetic acid (TFA), 30 mM SDS, and 300 mM LPS. All three peptides exhibited random coiled structures in sodium phosphate buffer. HPA3 (Fig. 12A) and HPA3P (Fig. 12B) both had a-helical structures in 50% TFA, 30 mM SDS and 300 mM LPS, while HPA3P2 (Fig. 12C) had a loose a-helical structure. 4. Discussion In the past, the discovery of new antibiotics was primarily serendipitous. Although the value of this approach is without question, little is learned from the successes and failures encountered in this process. Current antibiotics are mainly derived from natural sources and inhibit conserved macromolecular machinery such as that involved in DNA replication and protein or cell wall synthesis. This narrow set of targets is essential for cell multiplication, independent of the environment. With the advent of drug resistance, however, there is a need to move away from these antibiotics to new types of drugs. Among the new classes of molecules are the AMPs. Up to now, the reported studies of AMPs, including our own, have dealt with their isolation, characterization, possible

mechanisms of action and the effects of amino acid substitution. There has also been speculation about their degradation in vivo. In the present study, we report on a newly designed helix-PXXP-helix structure that is efficacious in mice infected with MDRPA, without exhibiting cytotoxicity, and also appears to down-regulate endotoxin-induced production of various pro-inflammatory cytokine and chemokines. HPA3 is an analog of HP (2-20), a peptide composed of residues 2e20 of H. pylori Ribosomal Protein L1. In an earlier report we found that HPA3 acts via a pore-forming mechanism to exert its potent antimicrobial effects [19]. However, it has never been tested in vivo due to its cytolytic effects at high concentrations. We also showed that adding a kink to the mid-region of HPA3 to form HPA3P changes the mechanism of action such that the molecule penetrates the cell membrane and binds to nucleic acids [21]. But this peptide is also not an ideal antibiotic in vivo. In the present study, we wanted to test the in vivo effects of an HPA3 analogue containing a PXXP motif. We therefore investigated the antibacterial effects of HPA3P2 both in vitro and in vivo. The helix-PXXP-helix structure, which contains two Pro residues and a flexible central hinge, was created by substituting Pro residues for the Glu and Phe residues at positions 9 and 12. Apparently, this structure is a key determinant of the hemolytic and cytotoxic properties of the peptide, as HPA3P2 exhibited potent antimicrobial activity against both drugsusceptible reference strains and multidrug-resistant bacterial strains, while its cytotoxicity in hRBCs and mammalian cells was remarkably low (Tables 1 and 2). In addition, the use of radial diffusion assays [38,39] enabled us to assess the antibacterial activities of these peptides against several clinical isolates of E. coli,

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Fig. 9. In vitro binding of HPA3P to plasmid DNA. (A) Gel retardation assay showing the DNA-binding activity of HPA3P (a) and buforin-2 (b); the peptide/DNA ratios are indicated at the top. (B) Trp blue shift assay of HPA3P binding to pDNA; the fluorescent signal was recorded using a fluorescence spectrometer (Em. 285 nm, Ex. 300e400 nm). (C) Transmission electron micrographs showing pDNA negatively stained with 1% (w/v) uranyl acetate (UAC). Bar ¼ 50 nm. The images were obtained in the absence of peptide (a) or in the presence of HPA3P (peptide/DNA: 0.5 w/w) (b) or (peptide/DNA: 1, w/w) (c).

P. aeruginosa, S. aureus and B. subtilis. This method is very sensitive and simple and has a good reproducibility. Using this assay system, we found that HPA3 and HPA3P had similar bactericidal effects against all four strains, while HPA3P2 exhibited particularly strong bactericidal effects against E. coli and P. aeruginosa (Fig. 1). We used an ICR mouse bacterial infection model to assess the in vivo activities of HPA3P2 against MDRPA. Notably, mice treated with single doses (0.5 mg/kg) and multiple doses (3  0.1 mg/kg) of HPA3P2 peptide showed 100% survival (Fig. 2A). And while the

histological analysis of lung tissue revealed significant hemorrhaging, disrupted alveolar tissue, and immune cell infiltration in untreated mice, tissues were almost completely protected in mice treated with 0.5 mg/kg HPA3P2 (Fig. 2B). This means that although the HPA3P and HPA3P2 exhibit similar cellular localizations their in vivo activities are very different. The outer membrane of Gram-negative bacteria contains LPS, which is secreted during cell division and is recognized by TLR4 on immune cells via interaction with LPS-binding protein

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Fig. 10. Inhibitory effects of HPA3P on expression of recombinant GFP. E. coli cells transfected with pET28a-GFP were incubated for 1 h in the absence of peptide or with HPA3P at its 1/4 or 1/2 MIC, after which 0.5 mM IPTG was added to induce expression of GFP for 3 h. (A) Phase contrast (top panels) and fluorescence (bottom panels) images of the GFP transfectants. (B) Western blot showing GFP expression was disrupted in E. coli incubated with 1/4 and 1/2 MIC of HPA3P.

(LBP) (Fig. 13A). Upon LPS recognition (e.g., in diseases such as pneumonitis), the NF-kB signaling pathway is activated, leading to the expression of various pro-inflammatory cytokines (TNF-a, IL-1b, and IL-6) and chemokines (CXCL5) [40]. We found that levels of signaling proteins (TLR4, NF-kB), pro-inflammatory cytokines (TNF-a, IL-1b, and IL-6) and CXCL5 chemokine were lower in lung tissue following treatment with 0.25 mg/ml HPA3P2 than in tissues from untreated mice. HPA3P2 apparently neutralized LPS on the membrane of MDRPA cells, thereby down-regulating expression of inflammatory signaling proteins, cytokines and chemokines (Fig. 13B). In addition, HPA3P2 reduced the numbers of GFP-expressing E. coli in the stomach, kidney and liver of ICR mice, indicating the peptide achieved good penetration of all three organs, enabling it to exert its bactericidal effect (Fig. 3).

HPA3P2 was able to effectively detoxify the liver. Liver damage induced by D-GalN is generally reflected by disruption of liver cell metabolism, which leads to characteristic changes in malondialdehyde (MDA) [41] and glutathione (GSH) levels in liver homogenates [42]. The mechanism underlying D-GalN-induced liver damage is thought to involve destabilization of cell membranes due to lipid peroxidation [43]. Consistent with that idea, we found that MDA levels were elevated in D-GalN-intoxicated rats, which is indicative of lipid peroxidation. It is therefore noteworthy that when we administered 500 mg/ml D-GalN and 16.7 mg/kg P. aeruginosa LPS to disrupt liver function and induce septic shock, ICR mice treated with peptide showed little to no damage to their liver and lungs. By contrast, untreated showed hemorrhaging, infiltration of immune cells and disruption of lung and liver tissues (Fig. 5).

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Fig. 11. In vitro anti-inflammatory effects of HPA3, HPA3P and HPA3P2. (A) LPS-neutralizing activities were determined in LAL assays. (B) Northern blot showing E. coli LPS-induced TNF-a gene transcription in RAW 264.7 cells. The treatment conditions are indicated at the top. (C), LPS-induced TNF-a secretion from RAW 264.7 cells. TNF-a levels were measured using an ELISA.

In our in vitro studies, HPA3 to be potently antibiotic but unacceptably cytotoxic, while HPA3P, with a single Pro substitution, remained potently antibiotic but much less cytotoxic. However, although HPA3P showed antibiotic activity in infected mice, it was ultimately not protective, and all HPA3P-treated mice died after a few days. With two Pro substitutions, the antibiotic activity of HPA3P2 was similar to that of HPA3P in vitro, but much greater in vivo. We think this likely reflects the different bacterial targets of the two peptides. It was previously shown that HPA3 acts against bacteria via a pore-forming mechanism [44]. In the present study,

we used rhodamine-labeled HPA3, HPA3P and HPA3P2 to show that although the three peptides are structurally similar, they act against E. coli cells through three different mechanisms of action: whereas Rho-HPA3 and Rho-HPA3P2 localized at the cell surface, HPA3P accumulated in the cytoplasm, suggesting it acts intracellularly. Consistent with that idea, SYTOX green uptake analysis showed that HPA3P penetrates into the cytoplasm in a manner similar to buforin-2, an AMP that targets the cytoplasm after permeating the cell membrane. Interestingly, HPA3P2 did not interact with or disrupt the inner membrane of Gram-negative bacteria (Fig. 7). It

Fig. 12. Secondary structures of HPA3 (A), HPA3P (B) and HPA3P2 (C) in aqueous solution and the indicated membrane-like environments. Far-UV CD spectra obtained under the indicated conditions are expressed as mean molar ellipticity (q). The peptide concentration was 30 mM.

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Fig. 13. Schematic diagram of the signaling pathway to LPS-induced septic shock in the absence (A) and presence (B) of HPA3P2. Abbreviations: LPS, lipopolysaccharide; LBP, LPSbinding protein; TLR4, toll-like receptor 4.

appears that the positions and number of Pro residues are a critical determinant of cellular localization and thus the mechanism of action of these peptides. Like buforin-2, HPA3P displayed strong binding affinity for pDNA (Fig. 9A). Our Trp blue shift assays showed peak HPA3P-pDNA binding at a 1:1 concentration ratio (Fig. 9B), while TEM showed corresponding pDNA aggregation (Fig. 9C). In addition, HPA3P dose-dependently reduced fluorescence levels in GFP-expressing E. coli, which suggests that after entering cells through selfformed pores, HPA3P inhibits protein synthesis via binding to DNA following penetration into cells.

On the other hand, HPA3P2 showed a stronger ability to neutralize P. aeruginosa LPS than the other peptides. The outer leaflet of the outer membrane consists of LPS, making it necessary for the peptide to bind to LPS and cross the outer membrane before entering the periplasmic space [45]. We found that HPA3P2 strongly bound LPS (Fig. 11A) and that this binding correlated with its ability to induce membrane destruction, suggesting it effectively disrupted the outer membrane and accessed the cytoplasmic membrane. At the same time, we know that in cases of septic shock, TNF-a is one of the first pro-inflammatory cytokines released from LPS-stimulated immune cells. Consistent with its ability to bind and

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neutralize LPS, HPA3P2 significantly reduced TNF-a gene transcription and translation in LPS-treated macrophages, as compared to cells treated with HPA3 or HPA3P (Fig. 11B and C). This suggests that unlike HPA3 or HPA3P, HPA3P2 has both potent antimicrobial and antiendotoxin activities. We analyzed the secondary structures of the peptides in various environments to determine the relationship between function and structure. We found that HPA3P showed less a-helicity than HPA3, and HPA3P2 showed a remarkable lack of a-helicity in TFA, SDS and LPS (Fig. 12). These conformational changes were brought about by interactions between the peptides and cell wall components (LPS). CD spectroscopy revealed that HPA3P2 undergoes major conformational changes when associating with LPS, and the ability to bind LPS is a prerequisite for its antibacterial and anti-endotoxin activities [46]. Sepsis is a major cause of mortality in intensive care units, accounting for 200,000 deaths every year in the United States alone [47]. Therefore, an effective AMP should not only exhibit prokaryotic selectivity, it should also have the ability to sequester LPS and ameliorate its toxicity. Recently, AMPs were reported to exert powerful effects against endotoxin shock by blocking the binding of LPS to CD14 þ cells, thereby suppressing the production of cytokines by these cells [48]. We have been endeavoring to develop AMPs with improved prokaryotic selectivity and greater LPS-neutralizing activity than HPA3, HPA3P or melittin. Our findings suggest HPA3P2 is uniquely active in vivo and warrants further detailed investigation into the detailed mode of action, including the role of its helix-PXXP-helix structure. Up to now, the translation of AMPs from in vitro experimentation to in vivo activity has up to now been difficult due to high systemic toxicity, instability, and inactivation and degradation in vivo. This study provides an example of an effective and safe newly designed AMP, and highlights the potential for unique AMPs to serve as effective antibiotics for use against multidrug-resistant bacterial strains. 5. Conclusion The discovery of new antibacterial drugs, effective against current multidrug-resistant strains, is essential to prevent a possible return to the pre-antibiotic era. Traditional antibiotics target essential cellular components and processes such as DNA synthesis, ribosomes and cell wall constituents, making these drugs effective mostly during bacterial growth. However, the ability of bacteria to reside in nature at other, more durable, stages and to express various inhibitors sets the need for alternatives to traditional antibiotics. In this report, we present an antimicrobial peptide, which we term HPA3P2. HPA3P2 targets the bacterial outer membrane, which is crucial for bacterial cell survival and resistance, and which synthesizes the signaling molecules required to activate the inflammatory proteins (e.g., cytokines and chemokines) that underlie septic shock. We found that not only does HPA3P2 cause bacterial cell death, it also inhibits the signaling molecules produced by Gram-negative bacteria. Further, HPA3P2 showed potent antibacterial activities against multidrug-resistant clinical isolates. These results suggest HPA3P2 could potentially serve as an effective pharmaceutical agent against multidrug-resistant bacteria. Acknowledgment This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011-0017532).

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