Special Issue Article Received: 24 December 2014

Revised: 12 March 2015

Accepted: 13 March 2015

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/psc.2781

Antimicrobial activities and action mechanism studies of transportan 10 and its analogues against multidrug-resistant bacteria‡ Junqiu Xie,§ Yuanmei Gou,§ Qian Zhao, Sisi Li, Wei Zhang, Jingjing Song, Lingyun Mou, Jingyi Li, Kairong Wang, Bangzhi Zhang, Wenle Yang and Rui Wang* The increased emergence of multidrug-resistant bacteria is perceived as a critical public health threat, creating an urgent need for the development of novel classes of antimicrobials. Cell-penetrating peptides that share common features with antimicrobial peptides have been found to have antimicrobial activity and are currently being considered as potential alternatives to antibiotics. Transportan 10 is a chimeric cell-penetrating peptide that has been reported to transport biologically relevant cargoes into mammalian cells and cause damage to microbial membranes. In this study, we designed a series of TP10 analogues and studied their structure-activity relationships. We first evaluated the antimicrobial activities of these compounds against multidrug-resistant bacteria, which are responsible for most nosocomial infections. Our results showed that several of these compounds had potent antimicrobial and biofilm-inhibiting activities. We also measured the toxicity of these compounds, finding that Lys substitution could increase the antimicrobial activity but significantly enhanced the cytotoxicity. Pro introduction could reduce the cytotoxicity but disrupted the helical structure, resulting in a loss of activity. In the mechanistic studies, TP10 killed bacteria by membrane-active and DNA-binding activities. In conclusion, TP10 and its analogues could be developed into promising antibiotic candidates for the treatment of infections caused by multidrug-resistant bacteria. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. Keywords: multidrug-resistant bacteria; cell-penetrating peptides; transportan 10; structure-activity relationships; action mechanism

Introduction Antimicrobial resistance is not a new problem but is a growing public health threat of broad concern to numerous countries and multiple sectors. A report from the World Health Organization has noted that a post-antibiotic era in which common infections and minor injuries can kill is far from being an apocalyptic fantasy; instead, this is a very real possibility for the 21st century [1]. According to the 2013 report of the National Antimicrobial Resistance Investigation Net (Mohnarin) from the Ministry of Health of the PRC, the Gram-positive bacteria Staphylococcus aureus (S. aureus) and Gram-negative bacteria Escherichia coli (E. coli), Klebsiella pneumonia (K. pneumonia), Pseudomonas aeruginosa (P. aeruginosa) and Acinetobacter baumannii (A. baumannii) had the highest separation rates among their respective types of bacteria. Among these, the drug resistance rate of E. coli was more than 50%. To help overcome the problem of antimicrobial resistance, we must race against time to develop new antibacterial agents. Cell-penetrating peptides (CPPs) were found to function as macromolecule carriers and as enhancers of cellular entry that act through distinct mechanisms. These properties were first discovered in HIV [2–4]. Because CPPs are short, cationic peptides that often have amphipathic properties and share features with antimicrobial peptides (AMPs) [5–7], certain studies have focused on whether CPPs have the same antimicrobial activities as AMPs do. Moreover CPPs show better distribution than AMPs do in vivo [8,9]. So CPPs are also expected to become a new generation of antibacterial substances. Transportan 10 (TP10) is a deletion analogue of the CPP transportan, a 21-residue chimeric construct

J. Pept. Sci. 2015

consisting of the N-terminal part of the neuropeptide galanin linked to the full-length wasp venom peptide mastoparan that lacks the toxic side effects of its parent peptide. TP10 can transport cargo across cell membranes [10,11] and it has been reported that TP10 can enter both mammalian and microbial cells, preferentially permeabilising and killing microbes such as S. aureus and Neisseria meningitides (N. meningitides) [6,9]. However, its potential use against multi-drug resistant bacteria with high separation rates when isolated from clinics has not been reported. It is also worth studying the structure-activity relationship of TP10. * Correspondence to: Rui Wang, Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Lanzhou University, 222 Tian Shui South Road, Lanzhou 730000, China. E-mail: [email protected] ‡

§

Special issue of contributions presented at the 13th Chinese International Peptide Symposium, Peptides: Treasure of Chemistry and Biology, June 30 - July 4, 2014 in Datong, Shanxi, China. Both author contributed equally to this work. Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Lanzhou University, Lanzhou, China Abbreviations: Aib, α-aminoisobutyric acid; AMPs, antimicrobial peptides; CLSI, Clinical and Laboratory Standards Institute; LB, Luria-Bertani; CPPs, cell-penetrating peptides; FITC, fluorescein isothiocyanate; Fmoc, N-9-fluorenylmethoxycarbonyl; IM, inner membrane; MH, Mueller-Hinton; NPN, 1-N-Phenylnaphthylamine; OM, outer membrane; ONPG, O-Nitrophenyl-β-D-galactoside; RP-HPLC, reverse-phase highpressure liquid chromatography; SEM, scanning electron microscopy; TE, Tris-EDTA; TFA, trifluoroacetic acid; TFE, trifluoroethanol; TP10, transportan 10; TSB, Trypticase Soy Broth.

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XIE ET AL. In the present study, TP10 and its analogues were designed and synthesised. Their antimicrobial activities against multidrugresistant bacteria were determined using the double dilution method. We investigated the effects of structure and charge on the antimicrobial activity of these compounds and further studied their abilities to inhibit S. aureus biofilm formation. Then, the cytotoxicity of TP10 and its analogues against mammalian cells was tested by haemolysis and the cell viability assay. The permeability of the E. coli outer and inner membranes to TP10 were determined by the NPN and ONPG assays. The cell membrane permeability was also observed using scanning electronic microscopy and laser confocal scanning microscope. Finally, the interaction between the peptides and DNA were examined using a binding assay.

were determined using the CLSI broth microdilution procedure [16] with certain modifications. Briefly, for MIC determinations, aqueous stock solutions containing all peptides were serially diluted twofold from 256 μg/ml to 0.25 μg/ml in MH broth. Then, 100 μl of each dilution was transferred into the wells of microplates (Costar 3599; Corning). A total of 100 μl of each bacterial suspension (1 × 106 CFU/ml) was then dispensed into the wells. A solution of MH broth alone served as a negative control. MICs were identified by a lack of turbidity after incubation at 37 °C for 18 h. All MIC determinations were made in triplicate, and if the results were within 2 doubling dilutions of each other, the highest reading was recorded for analysis. The experiment was repeated three times or more. Biofilm Inhibition Assay

Materials and Methods Peptide Synthesis All peptides were synthesised using a stepwise solid-phase method using N-9-fluorenylmethoxycarbonyl (Fmoc) chemistry, as reported previously [12]. The FITC was attached to the N-terminus via an aminohexanoic acid spacer by treating a resin-bound peptide (0.1 mmol) with FITC (0.1 mmol) and diisopropyl ethyl amine (0.5 mmol) in DMF for 12 h. All peptides were purified by a Sephadex gel column and RP-HPLC (Waters, MA, USA) with an XBridge BEH130 C18 column (10 μm, 4.6 × 250 mm) and a gradient elution of 5–95% acetonitrile with 0.1% TFA at a flow rate of 8 ml/min [13]. The atomic masses of the synthetic peptides were confirmed by electrospray ionisation-mass spectrometry. Bacteria and Reagents The bacterial strains used in this study were obtained from the Culture Collection Centre of School of Basic Medical Sciences of Lanzhou University (Lanzhou, China). All bacteria were cultured in LB broth. Before each assay was performed, the cells were grown overnight to the stationary phase at 37 °C in 5 ml LB broth. After incubation, 100 μl of bacteria was suspended in 5 ml of fresh LB broth (1/50 dilution) for an additional hour at 37 °C to obtain a mid-log-phase culture. NPN was purchased from J&K Scientific Ltd (Beijing, China) and ONPG was obtained from the Beyotime Institute of Biotechnology (Shanghai, China). A Bacteria Genomic DNA Kit was purchased from Tiangen Biotech (Beijing) Co., Ltd. Circular Dichroism Measurements Circular Dichroism (CD) experiments were performed using a JASCO J-810 spectropolarimeter (Tokyo, Japan) to investigate the secondary structures of all peptides. Samples were prepared by dissolving the peptides to the concentration of 100 μg/ml in one of the two solvents: 10 mM phosphate-buffered saline (PBS; pH 7.4) or 50% TFE/water (V/V) [14]. Measurements were performed in a cell with a 1 mm path length at room temperature. The following parameters were set: 50 nm/min scanning speed, 1 s response time and 1 nm bandwidth. For each spectrum (190–260 nm), the data of four scans were averaged and smoothed using the J-810 spectra analysis system [15]. Antimicrobial Assays The antimicrobial activities of all peptides were evaluated with the minimum inhibitory concentration (MIC). The MICs of all peptides

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Low concentrations of the peptides could inhibit the growth of biofilms. At sub-inhibitory concentrations, the capacity to inhibit biofilm formation was quantitatively measured [17,18]. S. aureus ATCC 25923 (5 × 106 CFU/ml) in TSB-glucose was incubated at 37 °C for 24 h in 96-well tissue culture microtitre plates with 200 μl of TP10, TP10-5 and TP10-7 at 1×, 1/2× and 1/4× MIC. Three wells were used for each peptide. After the incubation, the medium was discarded and washed twice with PBS to remove the non-adherent bacteria. Then, 200 μl of methanol was added per well for 5–10 min for fixation, and the plates were allowed to dry. The wells were stained with 200 μl of 0.1% crystal violet for 10 min. Later, the crystal violet solution from the plates was discarded, and the samples were thoroughly washed with distilled water 3 to 4 times and air dried at room temperature. The crystal violet stained biofilm was solubilised in 95% ethanol (200 μl) and the absorbance was recorded at 595 nm using a multimode reader (Tecan Infinite M200 PRO, Switzerland). The positive control was S. aureus in TSB-glucose without the peptide. Each assay was performed at least three times. Haemolytic Activity The ability of TP10 and its analogues to induce the haemolysis of human red blood cells was assessed as previously described with some modifications [19]. Red blood cells (4% haematocrit) were incubated for 1 h at 37 °C in PBS with a twofold serial dilution of all peptides from 4 μg/ml to 512 μg/ml. After centrifugation (1000 g), the absorbance of the supernatants was determined in a 96-well plate at 490 nm by using a Tecan Infinite M200 PRO. Date for 100% haemolysis was obtained by adding 0.1% Triton X-100. The negative control was PBS. The haemolysis rate of each peptide was calculated according to the following equation:
Haemolysis ð%Þ ¼ APeptide – APBS =ðATriton – APBS Þ  100%: The experiment was performed three times. Cytotoxicity Assay Cytotoxicity assays were performed using the human embryonic kidney cell line (HEK293A) and human umbilical vein endothelial cells (HUVECs). The cells were seeded into 96-well plates at 5 × 103 cells/well. After incubation for 24 h, the cells were treated for 24 h with increasing concentrations of a peptide (2 μg/ml to 512 μg/ml, in twofold increments) in triplicate. Then, 25 μl resazurin (1 mg/ml) was added to each well. After 4 h, the fluorescence was recorded by a multimode reader (Tecan Infinite M200 PRO). The excitation and emission wavelengths were 555 and 585 nm, respectively. Culture media with no peptides was regarded as 100% cell

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ANTIMICROBIAL ACTIVITIES OF TRANSPORTAN 10 AND ITS ANALOGUES survival, and the cell viability was calculated for each peptide. Each assay was performed at least three times.

OM Permeability The permeation of the E. coli OM by TP10 was determined using the NPN assay as previously described and with a minor modification [20,21]. E. coli ATCC 25922 cultures (1 × 108 CFU/ml) were centrifuged for 10 min at 1000 g. The cells were harvested, washed several times and resuspended in half the volume of 5 mM HEPES (pH 7.2). A total of 100 μl of E. coli cells and 50 μl of various concentrations of peptides were mixed with 50 μl of NPN (final concentration, 10 μM). As a negative control, 0.5% NaCl was added to the mixture of E. coli cells and NPN. Changes in fluorescence due to the partitioning of NPN into the OM were recorded as a function of time until no further increase in intensity was observed. The excitation and emission wavelengths were 350 and 420 nm, respectively. Each assay was performed at least three times using a multimode reader (Tecan Infinite M200 PRO).

IM Permeability The permeability of the IM was evaluated by the release of cytoplasmic β-galactosidase from E. coli into the culture medium using ONPG as the substrate [21,22]. E. coli ML-35 cells were cultured in LB broth. The cells (1 × 108 CFU/ml) were harvested, washed and suspended in 0.5% NaCl to an optical density of 1.2 at 420 nm, and then resuspended in half the volume of 0.5% NaCl solution. A total of 100 μl of E. coli and 90 μl of each of the various peptide concentrations were mixed with 10 μl of ONPG (30 mM) in a 96-well plate. The addition of 0.5% NaCl served as the negative controls. The o-nitrophenol production at 420 nm was determined using a Tecan Infinite M200 PRO multireader. Similar results were obtained after each assay was performed in triplicate.

Confocal Laser Scanning Fluorescence Microscopy E. coli ATCC 25922 cell suspensions were incubated at the mid-log phase and prepared with PBS buffer (1 × 108 CFU/ml). The mixture of cells and FITC-labelled TP10 (1× and 5× MIC) were incubated for 30, 60 min at room temperature. After incubation, the cells were washed with the same buffer three times and immobilised on a glass slide. The accumulation of the FITC-labelled peptide in the bacteria was observed with a 488 nm band-pass filter for the excitation of FITC by confocal laser scanning microscopy (Zeiss LSM 710 Meta).

SEM and Examination of the Bacterial Membrane E. coli ATCC 25922 cells at the mid-logarithmic phase were resuspended at a concentration of 1 × 108 CFU/ml in PBS and incubated with TP10 (final concentration: 1× and 5× MIC) at room temperature for 1 h. The mixture was then centrifuged for 5 min at 10 000 r.p.m. The control was prepared in the absence of peptides solution. The cells were fixed with 1 ml of 3% glutaraldehyde solution and were subsequently added into each tube. After fixation, the precipitates were incubated with 2.5% tannic acid for 2 days. Counter fixation in 2% osmium tetroxide for 1 h was followed by dehydration in ethanol and drying in a freeze-drying device (JFD-310; JEOL, Japan). The cells were coated with gold and analysed by SEM (JSM-6380Lv; Japan). Bacterial Genomic DNA-binding Assay E. coli ATCC 25922 genomic DNA was extracted using a TIANamp Bacteria DNA Kit. Gel retardation experiments of TP10 were performed as described previously [23,24]. Briefly, 10 μl (approximately 400 ng) of genomic DNA was dissolved in TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0) and mixed with an equal volume of varying concentrations of peptide (1×, 2×, 4× and 8× MIC). The reaction mixtures were incubated at room temperature for 30 min. Then, the mixtures and native loading buffer (10% Ficoll 400, 10 mM Tris–HCl, pH 7.5, 50 mM EDTA, 0.25% bromophenol blue and 0.25% xylene cyanol) were subjected to gel electrophoresis on a 1% agarose gel. The migration of DNA was detected by the fluorescence of ethidium bromide (Bio-Rad). The positive control included the AMP magainin 2, which was synthesised in our laboratory. The negative control did not include any of the peptides.

Results Peptide Design and Characteristics The sequences, hydrophobic ratios, net charges, masses and retention times of the synthesised TP10 and its analogues are summarised in Table 1. The sequences of TP10-2, TP10-3 and TP10-5 were reported previously [13]. Pro residues are prevalent in several AMPs, leading to changes in the secondary structures. Our previous results showed that the introduction of Pro decreased the cytotoxicity of TP10. According to the results previously mentioned, we designed TP10-7 and TP10-8. Because the retention time reflected the hydrophobic interactions between each peptide and the C18 stationary phase [25], we compared the hydrophobicity of the peptides by measuring the retention time on the C18 RP-HPLC column. As shown in Table 1, TP10-3 showed more

Table 1. Sequences, hydrophobic ratios, net charges, retention times of TP10 and its analogues Peptides TP10 TP10-2 TP10-3 TP10-5 TP10-7 TP10-8

Sequence

Hydrophobic ratioa

Net charge

Mass (Da)

tR (min)b

AGYLLGKINLKALAALAKKIL-NH2 AGYLLGKINLKPLAALAKKIL–NH2 AAYLLAKINLKALAALAKKIL-NH2 AGYLLGKINLKKLAKL(Aib)KKIL-NH2 AGYLLGKINLKPLAKL(Aib)KKIL-NH2 AGYLLPKINLKPLAKLPKKIL-NH2

61% 57% 71% nd nd 47%

5 5 5 7 6 6

2181.42 2207.44 2209.47 2309.56 2278.51 2330.54

19.787 19.194 19.909 18.847 19.180 16.089

a

Hydrophobic ratio and net charge were calculated by antimicrobial peptide database. tR (retention time) was measured by RP-HPLC.

b

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XIE ET AL. hydrophobicity than TP10 did, whereas the other analogues had less hydrophobicity. TP10-8 had the shortest retention time, meaning that it had the least hydrophobicity. This trend is consistent with the results calculated from the AMP database. CD Spectra To investigate the secondary structures of these peptides, the CD spectra for each peptide were measured in an aqueous environment (PBS) and a membrane-mimicking environment (50% TFE) [26]. As shown in Figure 1, TP10 showed a similar helical structure in PBS and a typical α-helical structure in 50% TFE solution. TP108, which included three Pro substitutions, showed a random coil structure and did not form a helical structure in the membranemimicking environment. The other peptides showed random coil structures in aqueous buffer while displaying the typical α-helical spectra, with two minimum peaks at 208 and 222 nm in 50% TFE solution. According to the molar ellipticity at 222 nm, TP10-3 showed the highest α-helical content of all the peptides. TP10-2

and TP10-5 had higher α-helicity compared with TP10. TP10-7 had significantly decreased α-helical contents. Antimicrobial Activity The antimicrobial activities of TP10 and its analogues were determined using the broth microdilution method. As shown in Table 2, TP10 had potent antimicrobial activity against all tested Gramnegative and Gram-positive bacteria, with MICs ranging from 8–32 μg/ml (3.67–14.67 μM). All analogues except TP10-8 were demonstrated to be active against both the standard strains and their drug-resistant counterparts. Notably, low concentrations (less than 4 μM) of TP10-5 and TP10-7 inhibited bacterial growth, especially against E. coli and A. baumannii. The results indicated that increasing the positive charge could enhance the antimicrobial activity. However, the antimicrobial activity of the peptide did not increase with increasing helical content (TP10-3). TP10-7, the peptide with the lower helical contents, had higher antibacterial activity. This activity was decreased and even lost with the

Figure 1. The CD spectra of TP10 and its analogues. The peptides are dissolved in 10 mM PBS (pH 7.4) (A) and 50% TFE/water (B), and the concentrations are fixed at 100 μg/ml. Data are expressed as the mean residue ellipticities.

Table 2. Antimicrobial activity of TP10 and its analogues MIC (μg/ml) (concentration in μM)

E. coli ATCC 25922 E. coli 780a E. coli 850a P. aeruginosa ATCC 27853 P. aeruginosa 2760a A. baumannii ATCC 19606 A. baumannii 2982a A. baumannii 651a A. baumannii 6138a K. pneumoniae ATCC 700603 S. aureus ATCC 25923 S. aureus 725a S. aureus 936a E. faecalis ATCC 29212 Bacillus subtilis ATCC 23857

TP10

TP10-2

TP10-3

TP10-5

TP10-7

TP10-8

Magainin 2

16 (7.33) 16 (7.33) 16 (7.33) 32 (14.67) 16 (7.33) 8 (3.67) 8 (3.67) 8 (3.67) 8 (3.67) 32 (14.67) 16 (7.33) 16 (7.33) 16 (7.33) 32 (14.67) 8 (3.67)

16 (7.25) 32 (14.5) 16 (7.25) 128 (57.99) 64 (28.99) 8 (3.62) 8 (3.62) 8 (3.62) 8 (3.62) 64 (28.99) 128 (57.99) 64 (28.99) >128 (57.99) >128 (57.99) 8 (3.62)

16 (7.24) 32 (14.48) 32 (14.48) 64 (28.97) 32 (14.48) 8 (3.62) 16 (7.24) 16 (7.24) 16 (7.24) 64 (28.97) 32 (14.48) 16 (7.24) 16 (7.24) >128(57.93) 8 (3.62)

8 (3.46) 8 (3.46) 8 (3.46) 16 (6.93) 8 (3.46) 8 (3.46) 8 (3.46) 8 (3.46) 8 (3.46) 16 (6.93) 16 (6.93) 16 (6.93) 16 (6.93) 32(13.86) 8 (3.46)

8 (3.51) 16 (7.02) 8 (3.51) 32 (14.04) 16 (7.02) 8 (3.51) 8 (3.51) 8 (3.51) 8 (3.51) 32 (14.04) 32 (14.04) 16(7.02) 32 (14.04) 32 (14.04) 8 (3.51)

>128 (54.92) >128 (54.92) >128 (54.92) 128 (54.92) 128 (54.92) 128 (54.92) 128 (54.92) 128 (54.92) 128 (54.92) >128 (54.92) >128 (54.92) >128 (54.92) >128 (54.92) >128 (54.92) 128 (54.92)

64 (25.95) 64 (25.95) 32 (12.97) 256 (103.80) >256 (103.80) 16 (6.49) 16 (6.49) 16 (6.49) 16 (6.49) >128 (51.90) >256 (103.80) >256 (103.80) >256 (103.80) >128 (51.90) 16 (6.49)

a

Multidrug-resistant bacteria isolated from clinics.

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ANTIMICROBIAL ACTIVITIES OF TRANSPORTAN 10 AND ITS ANALOGUES substitution of three Pro (TP10-8). Additionally, the antimicrobial activities of these peptides were compared with that of the AMP magainin 2, which has been studied more thoroughly. The results showed that all of the analogues, except TP10-8, had more potent activity against the bacteria studied than magainin 2 did. Some of the peptides had effective activities towards resistant bacteria, indicating that the peptides had a different mode of action on multidrug-resistant bacteria but not on bacteria with the conventional mechanism of resistance. Anti-biofilm Activity We tested TP10, TP10-5 and TP10-7, which had potent antimicrobial activities, for their anti-biofilm activities against S. aureus ATCC 25923 at sub-inhibitory concentrations using the crystal violet reporter assay. Figure 2 shows that the peptides demonstrated a dose-dependent inhibition of biofilm formation. TP10-7 was more effective than the others at inhibiting bacterial biofilm formation. The rate of biofilm production of S. aureus was 47.50%, even when the concentration of TP10-7 was 1/4 × MIC. In Vitro Toxicity Among the analogues, only TP10-5 had a higher haemolytic activity than TP10, whereas the others had lower haemolytic activities against human red blood cells (Figure 3A). The haemolytic activity of TP10 and its analogues was ranked in the following order: TP10-5 > TP10 > TP10-7 > TP10-3 > TP10-2 > TP10-8. TP10-5, which had the most positive charge, increased the haemolytic activity.

Figure 2. The inhibition of S. aureus ATCC 25923 biofilm formation by TP10 and its analogues. The control bars were S. aureus in TSB-glucose without peptide, set as 100%. Each assay was performed at least four times in triplicate. The results were expressed as percentage of biofilm formed with respect to control.

When 512 μg/ml of peptides were incubated for 1 h, TP10-7 and TP10-3 yielded 59.45% and 39.57% haemolysis, respectively. In contrast, even at concentrations of up to 512 μg/ml, TP10-2 and TP10-8 showed no haemolytic activity. The cytotoxicity of all of the peptides against mammalian cells was tested using the resazurin assay. The cytotoxicity of the peptides to HEK293A cells and HUVECs had the same order: TP10-5 > TP10-7 > TP10 > TP10-3 > TP10-2 > TP10-8 (Figure 3B and C). TP10-5 showed the highest cytotoxicity, particulary to HUVEC. Among the analogues, TP10-2, TP10-3 and TP10-8 had lower cytotoxicity than TP10 did. Even at concentrations of up to 512 μg/ml, TP10-8 showed no cytotoxicity against these two types of cells. Permeabilisation of the Outer and Inner Membranes To confirm whether TP10 had a similar mode of action as that of most AMPs, its effect on the integrity of the cell membrane including the permeability of the OM and IM to the peptide were measured. As shown in Figure 4, the addition of various concentrations of TP10 to an E. coli suspension in the presence of NPN caused a concentration-dependent increase in fluorescence, indicating that TP10 was capable of disrupting the OM of E. coli. It is worth noting that when NPN was incubated with the peptide-treated E. coli cells, the fluorescence intensity increased rapidly, reaching its maximum within a few minutes. Then, the permeation of ONPG into the cytoplasm was used to evaluate the TP10-induced permeabilisation of the E. coli IM. As seen in Figure 5, TP10 increased the absorbance value, indicating a marked release of β-galactosidase from the cell. The release reached a steady state

Figure 4. The effects of TP10 on the permeabilisation of the outer membrane of E. coli ATCC 25922. The peptide-mediated NPN uptake by E. 8 coli (1 × 10 CFU/ml) was measured by the intensity of NPN fluorescence. The data are the means ± standard errors of the means (n = 3).

Figure 3. The toxicity of TP10 and its analogues. (A) Haemolytic activity on human red blood cells. (B) The cytotoxicity to HEK293A. (C) The cytotoxicity to HUVECs. The data are the means ± standard errors of the means (n ≥ 3).

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XIE ET AL.

Figure 5. The effects of TP10 on the integrity of the inner membrane of E. coli ML-35. The release of cytoplasmic β-galactosidase from E. coli cells 8 (1 × 10 CFU/ml) treated with TP10. The data are the means ± standard errors of the means (n = 3).

at 30 min and the effect of TP10 on the integrity of the E. coli IM occurred in a time-dependent and concentration-dependent manners. Internalisation of FITC-labelled TP10 into E. coli Cells To determine the site of action of TP10, the FITC-labelled peptide was incubated with log-phase E. coli ATCC 25922 cells, and its localisation was visualised by confocal laser scanning microscopy. On the basis of the observed phenomenon, a subpopulation of the bacteria had intracellular fluorescence when incubated for 30 min (1 × and 5 × MIC) and 60 min at 1 × MIC (Figure 6A, B and C). When incubated for 60 min at 5 × MIC, the fluorescence had spread throughout the cells. This means that the localisation of TP10 was almost in the cytoplasm of the bacterial cells (Figure 6D), which was in striking contrast to the control (Figure 6E). With the increase of the incubation time and the concentration, more peptides had penetrated into the bacteria. This result illustrated that FITClabelled TP10 could penetrate the cell membrane of E. coli and accumulate in the cytoplasm, and that the mode of entrance was time-dependent and concentration-dependent. Morphological Changes in Bacteria by SEM The effect of TP10 on the morphology of the bacterial membrane was evaluated by SEM following treatment with the peptide at 1 × and 5 × MIC for 1 h. As illustrated in Figure 7, the membranes of the untreated control E. coli were normal and smooth (Figure 7A). In contrast, the treatment with peptides induced remarkable morphological changes. After treatment with TP10 at 1 × MIC, the E. coli developed a rough, ruffled surface (Figure 7B). At a high concentration, the E. coli became swollen. The membrane displayed blebbing and ruptured, and the intracellular contents had dispersed across the surface (Figure 7C). DNA-binding Activity As mentioned previously, TP10 can disturb the integrity of the bacterial membrane. However, whether TP10 is able to target the intracellular bacterial genome remains unknown. In the present study, we examined the DNA-binding activity of TP10 by analysing the electrophoretic mobilities of genomic DNA bands at various peptide/DNA weight ratios. Figure 8 indicates that TP10 could interact with E. coli genomic DNA and could retard its migration

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Figure 6. The internalisation of FITC-labelled TP10 into E. coli cells by 8 confocal laser scanning microscopy. E. coli ATCC 25922 (1 × 10 CFU/ml) were incubated with TP10 (A–D) and a PBS control (E) for 30 or 60 min at room temperature. (A) Cells treated with TP10 at 1× MIC for 30 min. (B) Cells treated with TP10 at 1× MIC for 60 min. (C) Cells treated with TP10 at 5× MIC for 30 min. (D) Cells treated with TP10 at 5× MIC for 60 min. (E) Untreated E. coli cells.

within the gels in a concentration-dependent manner. At a peptide concentration of 1 × MIC, some of the genomic DNA was still able to migrate into a gel like the nontreated DNA, whereas at a concentration of 2 × MIC, almost all of the DNA remained at the origin. At higher concentrations (4 × and 8 × MIC), complete retardation of DNA migration was observed. In contrast, the membrane-active antimicrobial peptide magainin 2 did not show any DNA-interacting ability even at concentrations of up to 64 μg/ml, a result that was absolutely different from the results for TP10. These results show that TP10 has an intrinsic ability to bind to DNA.

Discussion Hospital-acquired infections caused by multidrug-resistant bacteria, including E. coli, K. pneumonia, P. aeruginosa, A. baumannii and S. aureus, have progressively increased over the past decade [18]. Moreover, these important human pathogens possess increased resistances against almost all conventional antibiotics [27]. In recent years, many reports from the scientific community have shown that antibacterial drug development will not adequately address the problems posed by increasingly multidrug-resistant bacteria. The development of new antibacterial agents with activity against multi-drug resistant bacteria is therefore perceived as a critical public health need [28].

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ANTIMICROBIAL ACTIVITIES OF TRANSPORTAN 10 AND ITS ANALOGUES

8

Figure 7. The effect of TP10 on the bacterial membrane of E. coli ATCC 25922 by SEM. A sharp contrast can be seen between untreated E. coli (1×10 CFU/ml) and bacteria treated with TP10. (A) Control. (B) Cells treated with TP10 at 1 × MIC. (C) Cells treated with TP10 at 5 × MIC.

Figure 8. The interaction of TP10 with E. coli genomic DNA. The binding was assayed by measuring the inhibition of migration of the DNA bands. Various concentrations of TP10 were incubated with E. coli genomic DNA for 30 min at room temperature prior to electrophoresis on a 1% agarose gel. The control was genomic DNA without the peptides.

Cell-penetrating peptides have been previously shown to be powerful transport vector tools for the non-disruptive intracellular delivery of a large variety of cargoes through the cell membrane [4,9]. CPPs are a class of diverse peptides that are typically composed of 5–30 amino acids. Most of the known CPPs have net positive charges and are amphipathic. It is clear that CPPs share the common features with AMPs [29,30]. In line with this similarity, some CPPs exhibit antimicrobial activities against bacteria, parasites and fungi in vitro [31–33]. TP10 is a shorter analogue of the original transportan peptide that also has reduced toxicity. The N-termini of peptides from this chimeric family contains a sequence derived from the neuropeptide galanin, whereas the C-terminals contain a sequence from the wasp venom mastoparan. The two parts are linked by an extra Lys residue and the hybrid peptides are highly cationic [10,34–36]. TP10 not only could transport a variety of biologically relevant cargoes into mammalian cells but was also found to damage microbial membranes [6,9]. In the present study, TP10 and its analogues were first synthesised. Studies have shown that Aib-containing peptides nearly invariably adopt a helical backbone. The achiral amino acid Aib is a helix promoter/stabiliser and can markedly influence the properties of small bioactive peptides. Lys5, Lys8 and Aib10 MP (Aib-MP) is an analogue of mastoparan that is at the C-terminus of TP10. The increased charge and amphiphilicity of Aib-MP can enhance cellular penetration, protein binding or the activation of cellular targets or even a combination of these parameters [37]. Therefore, TP10-5 was previously designed to have an increased positive charge and a greater amphipathicity by substituting the C-terminal mastoparan

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with Lys5, Lys8 and Aib10. Previous data show that although TP10-5 has a stronger ability to penetrate cells, its cytotoxicity is also greatly increased [13]. In our study, TP10-5 had more potent antimicrobial activity than TP10 did. As in the previous results, the cytotoxicity was increased. However, TP10-2, the analogue with a Pro substitution at Ala12, had greatly reduced cytotoxicity in comparison with TP10. Based on the experimental results described previously, we designed a TP10-5 analogue named TP10-7 in which Lys12 was replaced with Pro to maintain efficient antimicrobial activity while reducing the cytotoxicity. Pro is singular among the 20 genetically encoded amino acids in terms of its rigidity, which is conferred by the pyrrolidine ring [38]. Pro-rich peptides stand out among AMPs and CPPs because they translocate across cellular membranes without inducing lysis or causing damage while also displaying much less toxicity to mammalian cells [39]. Thus, TP108, which contained three Pro substitutions, was designed to reduce the cytotoxicity. Our results showed that TP10 had potent antimicrobial activity against the tested bacteria. TP10-2, which had low toxicity, showed moderate activity. However, it was noticed that TP10-2 was effective against A. baumannii. TP10-5 and TP10-7 were found to have more potent antimicrobial activity, and TP10-7 had less toxicity. Unfortunately, TP10-8 had reduced toxicity but also lost its antimicrobial activity. TP10 is an amphipathic, cationic CPP, similar to helical AMPs [40]. The C-terminal region of TP10 derived from mastoparan is a welldefined α-helix, whereas the N-terminal galanin region is more disordered. The hinge between the two segments is located around Asn9 [36]. Similarly to AMPs, the antimicrobial activity of this type of CPP was affected by many factors, such as charge, amphipathicity and α-helicity. When peptides form α-helix structures, they will exhibit obvious amphipathicity. In general, the antimicrobial activity of peptides is improved by increasing the net charge and α-helicity [41]. Although TP10-3 had the highest helicity among these analogues, it showed low antimicrobial activity because of its lower positive charge. The introduction of Lys increased the helicity of TP10-5 as well as the number of positive charges. The increased helicity helped to maintain its antimicrobial activity, whereas the increased positive charge promoted the interaction of TP10-5 with the negatively charged bacterial cell membrane. Therefore, TP10-5 showed more potent antimicrobial activity against the tested bacteria. However, the higher helicity would lead to increased haemolytic activity. Therefore, TP10-5 had the highest toxicity against mammalian cells, such as HEK293A cells, HUVECs and human red blood cells. Pro substitution decreased the toxicity of the peptide TP10-2, as was shown previously [13]. The substitution of the TP10-5 Lys15 with Pro generated TP10-7, which had similarly effective antimicrobial activity and less toxicity compared with TP10-5. However, Pro is an amino acid that can disrupt the α-helical structure. The special side chain structure of Pro can change the secondary structure of the peptide chain, resulting in decreased

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XIE ET AL. helicity [42,43]. When three Pros were introduced into the peptide, the α-helix was destroyed. Thus, TP10-8 lost the ability to penetrate mammalian and bacterial cells. It was inferred that the spatial structure of the N-terminal glutathione in the chimeric peptide TP10 was crucial to its activity. Next, the membrane permeability of TP10 was studied. Our results revealed that the peptide can disrupt the integrity of both the OM and IM of E. coli. Similar to membrane-active AMPs, TP10 kills bacteria by disrupting the membranes. Although the mode of action of AMPs usually involves disrupting the integrity of the bacterial cytoplasmic membrane in many ways, other antimicrobial mechanisms have now been characterised in which the antimicrobial substance interacts with intracellular targets such as bacterial DNA, RNA and protein [44]. In this study, the DNA-binding assay showed that the migration of genomic DNA incubated with TP10 was retarded. It can be concluded that TP10 effectively kills bacteria not only by disrupting the bacterial membranes but also by rapidly binding to DNA. TP10 can pass into mammalian cells without causing membrane damage but can still kill microbial cells at certain concentrations. The detailed steps involved in internalisation remain controversial but have been shown to be dependent on peptide and membrane composition [9,45]. Bacterial membranes are generally more negatively charged than mammalian cell membranes because of their higher contents of anionic phospholipids and lipoteichoic acid or lipopolysaccharide in Gram-positive and Gram-negative bacteria, respectively. By contrast, zwitterionic phospholipids and cholesterol are the main constituents of mammalian cell membranes. Therefore, due to their cationic nature, TP10 and its analogues may preferentially bind to bacterial membranes by electrostatic attraction and subsequently enter cells. In the present study, we designed some analogues by changing the structural characteristics of TP10. However, the selectivity of TP10 remains to be improved. More novel analogues should be designed in a variety of ways to obtain more potent antimicrobial activity and peptides with lower toxicity. Antimicrobial peptides, which have a broad spectrum of antimicrobial activity and a novel membrane-active mode of action, are promising alternatives to conventional antibiotics and may help to overcome the problem of antimicrobial resistance [46,47]. However, as drugs, AMPs lack the localised immune cell deposition provided by secretory host cells. CPPs and AMPs share common features and antimicrobial mechanisms, and CPPs show more favourable in vivo distribution properties than AMPs. Thus, CPPs may offer a means for improving both the distribution of AMPs and microbial cell killing [9]. Our results showed that TP10 kills bacteria by disrupting the membrane and by binding to DNA. The double targets of cell membranes and intracellular DNA mean that the chances in the resistance are minimal, as such, changes would require complete alteration of the cell membrane or bypassing several biochemical pathways [24,48]. As a result, TP10 and its analogues make it difficult for bacteria to generate resistance, which may present a new strategy for defending against multidrugresistant bacteria. In conclusion, our findings indicate that TP10 and its analogues exhibit potent activities against multidrug-resistant strains that are responsible for most nosocomial infections. At low concentrations, these peptides are able to inhibit the formation of biofilms by S. aureus. By studying structure-activity relationships, we found that Lys substitution could increase the antimicrobial activity of TP10. However, increasing the positive charge and amphipathicity could significantly enhance the cytotoxicity. Pro introduction could

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reduce the cytotoxicity but Pro substitution at certain sites could disrupt the helical structure, resulting in lost activity. In the mechanistic studies, TP10 killed bacteria not only by disrupting the membrane but also by binding to DNA, which is not affected in known mechanisms of resistance. Therefore, TP10 and its analogues could be developed as promising antibiotic candidates for the treatment of infections caused by multidrug-resistant bacteria. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (no. 91213302, 81302798 and 81473095), the Key National S&T Program ‘Major New Drug Development’ of the Ministry of Science and Technology (2012ZX09504001-003), The Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT: IRT1137), the Research Fund for the Doctoral Program of Higher Education of China (20130211130005), Innovation Group in Gansu Province (1210RJIA002), and the Fundamental Research Funds for the Central Universities (lzujbky-2014-143).

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