Esculentin-2CHa: A host-defense peptide with differential cytotoxicity against bacteria, erythrocytes and tumor cells

Peptides 39 (2013) 95–102

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Esculentin-2CHa: A host-defense peptide with differential cytotoxicity against bacteria, erythrocytes and tumor cells Samir Attoub a , Milena Mechkarska b , Agnes Sonnevend c , Gordana Radosavljevic d , Ivan Jovanovic d , Miodrag L. Lukic d , J. Michael Conlon b,∗ a

Department of Pharmacology, College of Medicine and Health Sciences, United Arab Emirates University, 17666 Al-Ain, United Arab Emirates Department of Biochemistry, College of Medicine and Health Sciences, United Arab Emirates University, 17666 Al-Ain, United Arab Emirates c Department of Microbiology and Immunology, College of Medicine and Health Sciences, United Arab Emirates University, 17666 Al-Ain, United Arab Emirates d Center for Molecular Medicine, Faculty of Medicine, University of Kragujevac, Kragujevac, Serbia b

a r t i c l e

i n f o

Article history: Received 10 October 2012 Received in revised form 6 November 2012 Accepted 6 November 2012 Available online 16 November 2012 Keywords: Esculentin-2 Frog skin peptide Antimicrobial Immunomodulatory Hemolytic Anticancer

a b s t r a c t The host-defense peptide, esculentin-2CHa (GFSSIFRGVA10 KFASKGLGK D20 LAKLGVDLVA30 CKISKQC) shows potent (MIC ≤ 6 ␮M) growth inhibitory activity against clinical isolates of multidrug-resistant strains of Staphylococcus aureus, Acinetobacter baumannii, and Stenotrophomonas maltophilia and differential cytotoxic activity against human erythrocytes (LC50 = 150 ␮M) and human non-small cell lung adenocarcinoma A549 cells (LC50 = 10 ␮M). Esculentin-2CHa significantly (P < 0.01) stimulates the release of the anti-inflammatory cytokine IL-10 by mouse lymphoid cells and elevates its production after stimulation with concanavalin A and significantly (P < 0.05) stimulates TNF-␣ production by peritoneal macrophages. Effects on IL-6 and IL-1␤ production were not significant. Removal of the hydrophobic N-terminal hexapeptide (GFSSIF) from esculentin-2CHa results in abolition of growth inhibitory activity against S. aureus and cytotoxic activity against erythrocytes and A549 cells as well as a marked (≥16fold) reduction in potency against A. baumannii and S. maltophilia. The primary structure of esculentin-2 has been poorly conserved between frog species but evolutionary pressure has acted to maintain the hydrophobic character of this N-terminal hexapeptide sequence. Removal of the cyclic C-terminal domain (CKISKQC) and replacement of the Cys31 and Cys37 residues by serine resulted in appreciable decreases in cytotoxicity against all microorganisms and against mammalian cells. The more cationic [D20K, D27K] analog showed a modest increase in potency against all microorganisms (up to 4-fold) but a marked increase in cytotoxicity against erythrocytes (LC50 = 11 ␮M) and A549 cells (LC50 = 3 ␮M). © 2012 Elsevier Inc. All rights reserved.

1. Introduction The emergence in all regions of the world of strains of pathogenic bacteria and fungi with resistance to commonly used antibiotics constitutes a serious threat to public health and has become a cause of concern not only to physicians but to the general public. Treatment options against infections caused by multidrugresistant Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) [6,24] and multidrug-resistant Gram-negative pathogens such as Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumonia, and Stenotrophomonas maltophilia [22,45] are becoming increasingly limited. Although antibiotic resistance was first observed in

∗ Corresponding author at: Department of Biochemistry, Faculty by College of Medicine and Health Sciences, United Arab Emirates University, 17666 Al-Ain, United Arab Emirates. Tel.: +971 3 7137484; fax: +971 3 7672033. E-mail address: [email protected] (J.M. Conlon). 0196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2012.11.004

nosocomial isolates, new clones of MRSA and multidrug-resistant E. coli have emerged in the community causing infections in young, otherwise healthy people [19,36]. The problems posed by the emergence of multidrug resistance in the treatment of bacterial infections are also encountered in cancer chemotherapy with tumors becoming unresponsive to commonly used agents [8]. There is an urgent need for new types of drugs with appropriate pharmacokinetic and toxicological profiles that are active against multidrug- or pandrug-resistant microorganisms and/or against tumors that are unresponsive to commonly used anticancer agents. Cytotoxic peptides with potent antibacterial and antifungal activity and the ability to permeabilize mammalian cells play an important role in the system of innate immunity that constitutes the first-line defense against invading pathogens for a wide range of vertebrate and invertebrate species and may also be important in defense against predators. Such peptides are being considered as candidates for development into both anti-infective [49,50] and anti-cancer [26,32,41] drugs. There is no single mechanism

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by which peptides produce cell death but their action generally does not involve binding to a specific receptor or intracellular target rather a non-specific interaction with the cell membrane that results in its loss of integrity and ultimate disintegration [4]. Consequently, cytotoxic peptides are usually active against microorganisms that are resistant to currently licensed antibiotics due to their markedly different and highly destructive mode of action. Skin secretions from many species of Anura (frogs and toads) contain a wide range of biologically active compounds that have excited interest because of their potential for drug development. Of particular interest are the host-defense peptides with broadspectrum antibacterial and antifungal activities and the ability to permeabilize mammalian cells. Frogs belonging to the extensive family Ranidae (currently 347 recognized species [21]) are a rich source of such compounds and more than 300 antimicrobial peptides have been isolated from the skin secretions of frogs belonging to this family [10]. Anti-cancer activity has been studied in much less detail but brevinin-1BYa and its dicarba derivative from Rana boylii [27] and temporin-1CEa [46] from Rana chensinensis are active against a range of tumor cell lines. These peptides are not tumor-specific in their cytotoxic action but show >5-fold greater potency against tumor cells than against erythrocytes or fibroblasts. Esculentin-2 was first isolated from the skin of the Eurasian frog Rana esculenta [42] (now reclassified as a hybrid between Pelophylax lessonae and Pelophylax ridibundus [21]) but peptides with structural similarity have subsequently been identified in several other Eurasian [12,31,47] and North American species [2,3,12,13,15,17,25]. Esculentin-2 peptides generally exhibit broad-spectrum antimicrobial activity and low cytoxicity to human erythrocytes. However, little is known about structure–function relationships within this peptide family. Esculentin-2CHa was first isolated from skin secretions of the Chiricahua leopard frog, Lithobates chiricahuensis collected in southern Arizona [15]. The peptide shows potent growth-inhibitory activity (MIC ≤ 10 ␮M) against reference strains of the Gram-negative bacteria E. coli, P. aeruginosa, and Klebsiella pneumoniae and against the opportunistic yeast pathogen Candida albicans [15] and so represents a candidate for anti-infective drug development. This study investigates the growth-inhibitory activity of esculentin-2CHa and a range of fragments and analogs against well-characterized multidrug-resistant clinical isolates of A. baumannii, S. maltophilia, and S. aureus collected from patients in the Abu Dhabi region of the United Arab Emirates. Antimicrobial activity is compared with cytotoxic activity against freshly prepared human erythrocytes and human non-small cell lung adenocarcinoma A549 cells [20]. In view of the fact that many frog skin antimicrobial peptides have been shown to possess immunomodulatory activities [38], the ability of esculentin-2CHa to induce and modulate production of proinflammatory and immunoregulatory cytokines by appropriately stimulated mice mononuclear cells is also investigated.

2. Materials and methods 2.1. Peptide synthesis Esculentin-2CHa was purified from L. chiricahuensis skin secretions as previously described [15]. All fragments and analogs were supplied in crude form by GL Biochem Ltd (Shanghai, China). The peptides were purified to near homogeneity by reversedphase HPLC on a (2.2-cm × 25-cm) Vydac 218TP1022 (C-18) column (Grace, Deerfield, IL) equilibrated with acetonitrile/water/TFA (28.0/71.9/0.1, v/v/v) at a flow rate of 6 mL/min. The concentration of acetonitrile was raised to 56% (v/v) over 60 min using a linear gradient. Absorbance was measured at 214 nm and 280 nm and the major peak in the chromatogram was collected manually. The final purity of all peptides tested was >98% as determined by symmetrical peak shape and electrospray mass spectrometry. The primary structures of the peptides used in this study are shown in Table 1. 2.2. Antimicrobial assays Reference strains of S. aureus (ATCC 25923) and E. coli (ATCC 25726) were purchased from the American Type Culture Collection (Rockville, MD, USA). Clinical isolates of MRSA were obtained from hospitals in the Abu Dhabi region of the U.A.E. Characterization of the strains by multilocus sequence typing (MLST), staphylococcal cassette chromosome (SCCmec) typing, accessory gene regulator (agr) typing, Staphylococcus protein A (spa) typing, and toxin gene carriage has been described previously [44]. The strains were resistant to all ␤-lactam antibiotics tested and to a range of non␤-lactam antibiotics. Five independent A. baumannii strains isolated at four different hospitals in Abu Dhabi Emirate were included in the study. The origin of the strains has been described previously [14]. The bacteria were resistant to all antibiotics commonly used to treat Acinetobacter infections including cephalosporins, carbapenems, fluroquinolones, aminogylcosides but remained sensitive to tigecycline and colistin. The clonal lineage of the strains, determined by a multiplex PCR assay, is Euro clone I (NM8 and NM75), Euro clone II (NM35 and NM109) and non-typable (NM124). S. maltophilia strains (B32/1, B32/4, B5/5, B6/2, U8708) were isolated from individual patients with bloodstream infection in Tawam Hospital, Al Ain, UAE [30]. The isolates exhibited different molecular fingerprints by pulsed-field gel electrophoresis. All strains were resistant to meropenem (MIC > 32 mg/L) but were sensitive to co-trimoxazole (MIC < 5 mg/L). Minimum inhibitory concentrations (MICs) of the peptides were measured in the concentration range of 0.75–200 ␮M by standard double dilution methods [9] and were taken as the lowest concentration of peptide where no visible growth was observed. The values were confirmed by measurement of absorbance at 630 nm. In order to monitor the validity and reproducibility of the assays, incubations with reference strains of bacteria were carried out in

Table 1 Physicochemical properties of esculentin-2CHa and its analogs.

Esculentin-2CHa [D20K,D27K] [C31S, C37S] (1–30) (7–37) (11–37) (15–37) (16–37)

Primary structure

Charge at pH7

Isoelectric point

␣-helicity

GRAVY

GFSSIFRGVAKFASKGLGKDLAKLGVDLVA CKISKQC GFSSIFRGVAKFASKGLGKKLAKLGVKLVA CKISKQC GFSSIFRGVAKFASKGLGKDLAKLGVDLVA SKISKQS GFSSIFRGVAKFASKGLGKDLAKLGVDLVA RGVAKFASKGLGKDLAKLGVDLVACKISKQC KFASKGLGKDLAKLGVDLVACKISKQC KGLGKDLAKLGVDLVACKISKQC GLGKDLAKLGVDLVACKISKQC

+5

10.82

17–33

−0.122

+9

11.97

17–24

−0.109

+5

10.82

17–24

−0.063

+3 +5 +4 +3 +2

10.55 10.82 10.54 10.38 10.16

17–24 11–27 7–23 5–19 4–18

−0.087 −0.220 −0.213 −0.243 −0.210

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parallel with increasing concentrations of ampicillin as previously described [14].

97

Calculated grand average of hydrophobicity (GRAVY) of the peptides was based upon the hydrophobicity scales for amino acids of Wimley and White [48].

2.3. Cytotoxicity assays 2.6. Statistical analysis The A549 cell line is derived from a human lung adenocarcinoma and has been used extensively as a Type II pulmonary epithelial cell model in drug metabolism and other studies [20]. A549 cells were maintained at 37 ◦ C in RPMI 1640 medium containing 2 mM l-glutamine and supplemented with 10% fetal bovine serum (FBS, Biowest, Nouaille, France), and antibiotics (penicillin 50 U/mL; streptomycin 50 ␮g/mL). In all experiments, cell viability was higher than 99% using trypan blue dye exclusion. Cells were seeded in 96-well plates at a density of 5 × 103 cells/well. After 24 h, cells were treated for 24 h with increasing concentrations of esculentin-2CHa and its analogs (1–100 ␮M) in triplicate. Control cultures were treated with 0.1% DMSO. The effect of the peptides on cell viability was determined by measurement of ATP concentrations using a CellTiter-Glo Luminescent Cell Viability assay (Promega Corporation, Madison, WI). Luminescent signals were measured using a GLOMAX Luminometer system. The LC50 value was taken as the mean concentration of peptide producing 50% cell death in three independent experiments. In order to determine the rate at which [K20, K27] esculentin-2CHa (3 ␮M and 10 ␮M) produced cell death, cell viability was measured after 30 min, 2 h, 6 h, and 24 h. Peptides in the concentration range 3–200 ␮M were incubated with washed human erythrocytes (2 × 107 cells) from a healthy donor in Dulbecco’s phosphate-buffered saline, pH 7.4 (100 ␮L) for 1 h at 37 ◦ C. After centrifugation (12,000 × g for 15 s), the absorbance at 450 nm of the supernatant was measured. A parallel incubation in the presence of 1% (v/v) Tween-20 was carried out to determine the absorbance associated with 100% hemolysis. The LC50 value was taken as the mean concentration of peptide producing 50% hemolysis in three independent experiments.

Statistical analyses were performed using commercially available software (SPSS version 13.0; SPSS Inc., Chicago, IL, USA). The distributions of data were evaluated for normality using Kolmogorov–Smirnov test and then retested with Chi-Square test. Comparison of quantitative parametric data between two study groups was done by application of unpaired t-test. Differences between the paired data were evaluated using the paired t-test. A P-value < 0.05, from two-sided tests, was considered statistically significant. 3. Results 3.1. Physicochemical properties of the peptides Secondary structure prediction based on helix/coil transition theory [34] indicates that esculentin-2CHa has a high probability of forming an extended ␣-helical conformation in the central to C-terminal region (residues 17–33) of the peptide (Table 1). An Schiffer–Edmundsen wheel [40] representation of this domain illustrates the amphipathic nature of the helix (Fig. 1). Replacement of the Asp20 and Asp27 residues by l-lysine generates a more cationic analog (charge of +9 at pH 7) with a shorter ␣-helical conformation (residues 17–24). Removal of the cyclic domain at the C-terminus of the peptide, either by deletion or replacement of the Cys31 and Cys37 residues by the isosteric l-serine, results in an analog in which helicity is also decreased. Progressive deletion of peptide domains from the N-terminus of esculentin-2CHa generates fragments in which an extended ␣-helical conformation is preserved but the peptides are less hydrophobic and less cationic than the naturally occurring peptide.

2.4. Measurement of cytokine release 3.2. Antimicrobial and cytotoxic activities of the peptides All chemicals were supplied by Sigma (Germany). Effects of esculentin-2CHa on cytokine production by mouse mononuclear cells were determined using previously described methodologies [16,28,38]. The peptide (20 ␮g/ml) was incubated with peritoneal macrophages of BALB/c mice (2 × 105 cells/well), with and without lipopolysaccharide (5 ␮g/ml)) and with lymphoid cells from mouse spleen (2 × 105 cells/well), with and without the T cell mitogen, concanavalin A (5 ␮g/ml). Cells were cultured for 24 h at 37 ◦ C in supplemented culture medium (RPMI 1640 containing 10% autologous serum, 2 mM L-glutamine, 100 IU/ml penicillin G and 100 ␮g/ml streptomycin) in a humidified atmosphere of 5% CO2 – 95% air. Cell number and viability were determined using trypan blue and acridine orange/ethidium bromide staining. After incubation cells were centrifuged, supernatants collected and kept at −20 ◦ C until time of assay. Concentrations of tumor necrosis factor␣ (TNF-␣), IL-1␤, IL-6, and IL-10 were determined using ELISA assay kits from R & D Systems (Minneapolis, USA) according to manufacturer’s recommended protocols.

The abilities of esculentin-2CHa and its analogs to inhibit the growth of reference strains of the Gram-positive bacterium S. aureus and the Gram-negative bacterium E. coli are compared with their hemolytic activities against human erythrocytes (Table 2) and

2.5. Secondary structure prediction Prediction of secondary structure and determination of % helicity per residue for the peptides were performed using the AGADIR program [34]. AGADIR is a prediction algorithm based on the helix/coil transition theory, which predicts the helical behavior of monomeric peptides. Calculations were performed at pH 7 and 278 ◦ K and at an ionic strength of 0.1. A minimum percentage of 1% helicity/residue was considered to predict the presence of a helix.

Fig. 1. Schiffer–Edmundson wheel representation of the predicted ␣-helical domain (residues 17–33) of esculentin-2CHa demonstrating the amphipathic nature of the conformation. The arrows denote the sites of replacement of amino acids by l-lysine in order to produce an analog with greater cationicity and cytotoxicity.

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Table 2 Minimum inhibitory concentrations (␮M) of esculentin-2CHa and its analogs against reference strains of microorganisms and cytotoxicities (LC50 values; ␮M) against human erythrocytes (RBC) and A549 cells.

Esculentin-2CHa [D20K, D27K] [C31S, C37S] [1–30] [7–37] [11–37] [15–37] [16–37]

S. aureus

E. coli

RBC

A549

6 3 25 100 >200 >200 >200 >200

6 6 50 > 100 100 >200 >200 >200

150 11 >200 >200 >200a >200a >200a >200a

10 3 26 ND >100b ND ND ND

ND: not determined. a No detectable hemolytic activity at 200 ␮M. b No detectable cytotoxic activity at 100 ␮M.

their cytotoxicities against human non-small cell lung adenocarcinoma A549 cells (Fig. 2). The abilities of the peptides to inhibit the growth of well-characterized, multidrug-resistant clinical isolates of S. aureus, and the opportunistic pathogens A. baumannii, and S. maltophilia are shown in Tables 3–5. Removal of the hydrophobic N-terminal hexapeptide (GFSSIF) from esculentin-2CHa results in abolition of growth inhibitory activity against S. aureus and cytotoxic activity against erythrocytes and A549 cells as well as a marked (≥16-fold) reduction in potency against A. baumannii and S. maltophilia. Similarly, removal of the cyclic C-terminal domain (CKISKQC) resulted in appreciable decreases in potency against all microorganisms. Replacement of

Table 3 Minimum inhibitory concentrations (␮M) of esculentin-2CHa against clinical isolates of multidrug-resistant Staphylococcus aureus.

Esculentin-2CHa [D20K, D27K] [C31S, C37S] [1–30] [7–37] [11–37] [15–37] [16–37]

127/08

145/08

274/08

V4180

S908

T4/6

6 6 50 >200 >200 >200 >200 >200

6 6 100 >200 >200 >200 >200 >200

6 6 100 >200 >200 >200 >200 >200

6 3 50 >200 >200 >200 >200 >200

6 6 100 >200 >200 >200 >200 >200

6 5 100 >200 >200 >200 >200 >200

Table 4 Minimum inhibitory concentrations (␮M) of esculentin-2CHa and its analogs against clinical isolates of multidrug-resistant Acinetobacter baumannii.

Esculentin-2CHa [K20, K27] [C31S, C37S] [1–30] [7–37] [11–37] [15–37] [16–37]

NM8

NM35

NM75

NM109

NM124

6 3 12.5 50 100 200 >200 200

6 1.5 12.5 50 200 200 >200 >200

6 3 12.5 50 100 200 >200 200

6 3 12.5 50 100 200 >200 200

6 3 12.5 50 50 100 >200 200

the Cys31 and Cys37 residues in this cyclic domain by l-serine produced a greater reduction in potency against S. aureus (≥8-fold) than against A. baumannii (2-fold) and S. maltophilia (≤4-fold). The more cationic [D20K, D27K] analog showed a modest increase in

Fig. 2. Effects of (A) esculentin-2CHa, (B) esculentin-2CHa-(7-37), (C) [C30S,C37S] esculentin-2CHa and (D) [D20K, D27K] esculentin-2CHa on the viability of A549 lung adenocarcinoma cells after 24 h exposure. All experiments were repeated at least three times. Columns: mean; bars: SEM.

S. Attoub et al. / Peptides 39 (2013) 95–102

Esculentin-2CHa [D20K, D27K [C31S, C37S] [7–37] [11–37] [15–37] [16–37]

B32/1

B32/4

B5/5

B6/2

U8708

6 3 12.5 100 100 >200 >200

3 1.5 6 50 50 200 200

6 1.5 12.5 100 100 >200 >200

3 3 6 50 100 >200 200

6 3 6 100 100 >200 >200

[Lys20,Lys27] Esculentin-2CHa

120

Cell viability (% of control)

Table 5 Minimum inhibitory concentrations (␮M) of esculentin-2CHa and its analogs against clinical isolates of multidrug-resistant Stenotrophomonas maltophilia.

99

100 3µM

80

10µM

60 40 20

potency against all microorganisms (up to 4-fold) but a 14-fold increase in cytotoxicity against erythrocytes (LC50 = 11 ␮M) and 3-fold increase against A549 cells (LC50 = 3 ␮M). Kinetic studies showed that the analog at a concentration of 10 ␮M produced >90% death of A549 cells after 30 min and 100% cell death after 6 h (Fig. 3). 3.3. Immunomodulatory actions of esculentin-2CHa At the concentration tested (20 ␮g/ml), esculentin-2CHa did not affect the viability of mononuclear cells (data not shown). The ability of esculentin-2CHa to modulate the production of proinflammatoty cytokines was tested in cultures of peritoneal macrophages. The peptide significantly (P < 0.05) stimulated TNF-␣ production by unstimulated cells but did not alter production in LPS-stimulated

0 0

0.5

2

6

24

Time (h) Fig. 3. Effect of [D20K, D27K] esculentin-2CHa (3 ␮M and 10 ␮M] on the viability of A549 lung adenocarcinoma cells as a function of time. All experiments were repeated at least three times. Columns: mean; bars: SEM.

cells (Fig. 4A). Production of IL-1 ␤ (Fig. 4B) and IL-6 (Fig. 4C) in either unstimulated or LPS-stimulated cells was not significantly altered. In order to gain insight into possible immunoregulatory actions on lymphoid cells, the effects of esculentin-2CHa on production of the Th2/Treg cytokine, IL-10 was investigated. There was

Fig. 4. Effects of esculentin-2CHa (20 ␮g/ml) on the production of (A) tumor necrosis factor-␣ (TNF-␣), (B) IL-1␤, and (C) IL-6 from unstimulated and lipopolysaccharide (LPS)stimulated mouse peritoneal macrophages and (D) IL-10 from unstimulated and concanavalin A (ConA)-stimulated mouse mouse lymphoid cells. Med refers to incubation with medium only.

100

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Rana chiricahuensis

GFSSIFRGVA KFASKGLGKD LAKLGVDLVA CKISKQC

Rana berlandieri

GLFSILRGAA KFASKGLGKD LTKLGVDLVA CKISKQC

Rana pretiosa

GVFSFLKTGA KLLGSTLLKM AGKAGAEHLA CKATNQC

Rana pretiosa

GIFSALAAGV KLLGNTLFKM AGKAGAEHLA CKATNQC

Rana esculenta

GILSLVKGVA KLAGKGLAKE GGKFGLELIA CKIAKQC

Rana esculenta

GIFSLVKGAA KLAGKGLAKE GGKFGLELIA CKIAKQC

Lithobates sevosus

GFFSLIKGVA KIATKGLAKN LGKMGLDLVG CKISKEC

Lithobates palustris

GLFSILKGVG KIALKGLAKN MGKMGLDLVS CKISKEC

Lithobates warzsewitschii

NIFSLLSLGA KVLGKTLLKS AGKAGAEQLA CKATNQC

Odorrana hosii

GIFSLIKGAA QLIGKTVAKE AGKTGLELMA CKVTKQC

Odorrana ishikawae

GIFSLIKGAA KLITKTVAKE AGKTGLELMA CKVTNQC

Hylarana erythraea

GLFSLFKAGA KILGKTFLKQ AGKAGAEHLA CKAANQC

Amolops loloensis

GIFALIKTAA KFVGKNLLKQ AGKAGLEHLA CKANNQC

Amolops loloensis

GIFSLIKTAA KFVGKNLLKQ AGKAGVEHLA CKANNQC

Fig. 5. A comparison of the primary structures of esculentin-2 from different frog species belonging to genera within the family Ranidae. Conserved residues are shaded, The N-terminal hexapeptide sequence is underlined. Rana esculenta has been reclassified as a Pelophylax lessonae × Pelophylax ridibundus hybrid [21].

significant increase (P < 0.01) of IL-10 production both in presence or absence of the T cell mitogen, concanavalin A (Fig. 4D). 4. Discussion Esculentin-2CHa combines potent antibacterial activity against multidrug-resistant clinical isolates of S. aureus, A. baumannii, and S. maltophilia with low cytotoxicity against human erythrocytes but appreciably higher (15-fold) cytotoxicity against human nonsmall cell lung adenocarcinoma A549 cells. The therapeutic index of the peptide, defined as the ratio of LC50 for erythrocytes to the MIC for bacteria, is between 25 and 50 for these microorganisms. This value compares favorably with other frog skin peptides or analogs based upon their structure. For example, a recent structureactivity study of alyteserin-2a, a peptide first isolated from the midwife toad Alytes obstetricans, led to development of the [S7k, G11k] analog (k = d-lysine) with a therapeutic index of 23 against A. baumannii, and S. maltophilia [14]. Similarly, the analog [G11k, N15K] alyteserin-2a is 13-fold more potent against A549 cells than against erythrocytes [16]. Preliminary analysis of the effects of esculentin-2CHa on immunoregulatory cytokine production (Fig. 5) suggests that, by stimulating production of TNF-␣, the peptide may have an enhancing effect in the innate immune response to microbial infection and tumorigenesis while acquired immunity may be downregulated by the production of IL-10. This scenario could be beneficial in a clinical setting in which the first line of defense should be enhanced without possible complications due to induction of autoimmunity. The relative potencies of membrane-permeabilizing peptides against bacteria and against mammalian cells are determined by complex interactions between cationicity, hydrophobicity, conformation (␣-helicity), and amphipathicity [11]. These parameters are not independent variables so that alteration of peptide structure by a selected amino acid substitution may change several physicochemical properties simultaneously. In contrast to esculentin-1b, a 46-amino-acid antimicrobial peptide from R. esculenta whose (1–18) fragment retains the high potency and broad spectrum activity of the intact peptide [33], the complete sequence of esculentin-2CHa is needed for high antimicrobial activity. The N-terminal hexapeptide (Gly-Phe-Ser-Ser-Ile-Phe) is identified as a domain that is of critical importance in determining both antimicrobial and cytotoxic activity. The (7–37) fragment of

esculentin-2CHa possesses the same cationicity and predicted degree of ␣-helicity as the native peptide but lacks cytotoxicity against mammalian cells and growth-inhibitory activity against a reference strain (Table 2) and multidrug-resistant clinical isolates (Table 3) of S. aureus. Studies with both naturally occurring and model peptides have shown that increasing hydrophobicity correlates with stronger hemolytic activity whereas there is an optimum range of hydrophobicity in which high antimicrobial activity is obtained [7,18]. Decreasing or increasing hydrophobicity beyond this window dramatically decreases antimicrobial activity. Consistent with these observations, removal of residues (1–6) from esculentin-2CHa decreases hydrophobicity appreciably (Table 1) which has the effect of abolishing or appreciably decreasing antimicrobial activity. Unlike the temporin, brevinin-1 and ranatuerin-2 families of antimicrobial peptides, which are found in skin secretions of a wide range of ranid frogs of both North American and Eurasian origin [10], the species distribution of esculentin-2 is sporadic. As shown in Fig. 5, orthologs has been isolated from Rana berlandieri [23], R. esculenta [42], Rana pretiosa [13], Lithobates palustris [3], Lithobates sevosus [25], Lithobates warzsewitschii [17], Odorrana hosii [12], Odorrana ishikawae [31], Hylarana erythraea [2], and Amolops loloensis [47]. The primary structure of esculentin-2 has been poorly conserved during the radiation of these genera with Lys19 , Cys31 , Lys32 , and Cys37 being the only invariant residues. However, evolutionary pressure has acted to conserve the hydrophobic character of the N-terminal hexapeptide (underlined in Fig. 5). This domain contains combinations of either three or four hydrophobic Phe, Leu, Ile and Val residues. Removal of the C-terminal cyclic domain (Cys-Lys-Ile-Ser-LysGln-Cys) from esculentin-2CHa generates an analog with both decreased cationicity and helicity (Table 1) that shows markedly reduced potency against S. aureus and A. baumannii (Tables 2–4). Studies with a range of amphipathic ␣-helical peptides (reviewed in [11,14]) have demonstrated that antimicrobial potency correlates with the positive charge on polar face of the helix until a limit is reached whereupon further increase in cationicity does not result in any further increase in activity. The decrease in antimicrobial potency of the (1–30) fragment is probably due, at least in part, to reduced cationicity (charge at pH 7 = +3 compared with +5 for esculentin-2CHa) but reduced helicity is also important. The justification for this statement is the observation that the acyclic [C31S, C37S] analog, which has the same cationicity as the native peptide but reduced helicity, also displays diminished antimicrobial and cytotoxic potency. This result is consistent with the report that the acyclic [C18S, C24S] derivative of brevinin-1BYa showed reduced antimicrobial and hemolytic activity compared with the naturally occurring peptide [27]. The [D20K, D27K] analog, with a molecular charge at pH 7 of +9 compared with +5 for the native peptide, shows the expected, albeit relatively modest, increase in antimicrobial potency (up to 4-fold) but a marked increase in hemolytic activity (14-fold). This result is reminiscent of a study in which increasing one positive charge on the polar face of the 26-residue amphipathic ␣-helical model peptide V13K from +8 to +9 resulted in a >32-fold increase in hemolytic activity [29]. As shown in Fig. 1, the degree of amphipathicity of esculentin-2CHa is relatively low because of the presence of the Lys32 residue on the hydrophobic face of the helix. In the analog, Lys19 , Lys20 , Lys23 , and Lys27 residues segregate together on the hydrophilic face of the helix with a polar angle (angle subtended by the charged residues) of 120◦ . Increases in amphipathicity correlate with increased hemolytic activity and small increases in the hydrophobic moment (a semi-quantitative measure of the amphipathicity of a ␣-helical peptide) may produce major changes in activity [18,29]. On the Wimley and White hydrophobicity scale [48], lysine (−0.99) is more hydrophobic than aspartic acid (−1.23)

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so that increased hydrophobicity along with increased cationicity and amphipathicity may also contribute for the greater hemolytic activity of the analog. Acidic amino acid residues are not uncommon in frog skin antimicrobial peptides and it is tempting to speculate that they are present to attenuate the cytotoxicity of the peptide so that it does not damage the frog’s own skin when released into secretions. Although effective new types of antibiotics against multidrugresistant Gram-positive bacteria such as MRSA have been introduced or are in clinical trials [43], the situation regarding new treatment options for infections produced by multidrug-resistant Gram-negative pathogens is less encouraging [22,45]. There has been a dramatic increase in the number of hospital-acquired infections caused by the opportunistic Gram-negative pathogens A. baumannii [35,36] and S. maltophilia [1,5,39] during the past decade. These are typically encountered in immunocompromised and critically ill patients in intensive care and burns units and also as a co-colonizer with P. aeruginosa in respiratory tracts of cystic fibrosis patients. Antibiotic resistance in these bacteria arises from production of ␤-lactamases and aminoglycoside-inactivating enzymes, activation of multi-drug efflux pumps, modification of the drug target, and poor penetration due to loss of porin(s). These mechanisms are unlikely to reduce the efficacy of antimicrobial peptides. Development of resistance to antimicrobial peptides has been demonstrated experimentally in vitro but it occurs at rates that are orders of magnitude lower than those observed for conventional antibiotics [37]. Further studies are warranted to assess the therapeutic potential of peptides derived from esculentin-2CHa as anti-infective agents. Although the potent anticancer peptide [D20K, D27K] esculentin-2CHa is too hemolytic for systemic application, appropriate amino acid substitutions by d-lysine instead of l-lysine will generate analogs with increased cationicity but reduced helicity that are expected to be less cytotoxic to erythrocytes (reviewed in [11,16]). Acknowledgments This work was supported by a Faculty Support Grant and a University Research Grant (G00000900) from U.A.E. University and by a grant from the Terry Fox Fund for Cancer Research. The authors thank Manju Prajeep, Kholoud Arafat, and Hama Arafat for technical assistance. The authors thank Prof. P.R. Flatt, University of Ulster, U.K. for a gift of peptides. References [1] Abbott IJ, Slavin MA, Turnidge JD, Thursky KA, Worth LJ. Stenotrophomonas maltophilia: emerging disease patterns and challenges for treatment. Expert Rev Anti Infect Ther 2011;9:471–88. [2] Al-Ghaferi N, Kolodziejek J, Nowotny N, Coquet L, Jouenne T, Leprince J, et al. Antimicrobial peptides from the skin secretions of the South-East Asian frog Hylarana erythraea (Ranidae). Peptides 2010;31:548–54. [3] Basir YJ, Knoop FC, Dulka J, Conlon JM. Multiple antimicrobial peptides and peptides related to bradykinin and neuromedin N isolated from skin secretions of the pickerel frog, Rana palustris. Biochim Biophys Acta 2000;1543:95–105. [4] Bocchinfuso G, Palleschi A, Orioni B, Grande G, Formaggio F, Toniolo C, et al. Different mechanisms of action of antimicrobial peptides: insights from fluorescence spectroscopy experiments and molecular dynamics simulations. J Pept Sci 2009;15:550–8. [5] Brooke JS. Stenotrophomonas maltophilia: an emerging global opportunistic pathogen. Clin Microbiol Rev 2012;25:2–41. [6] Chambers HF, Deleo FR. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat Rev Microbiol 2009;7:629–41. [7] Chen Y, Guarnieri MT, Vasil AI, Vasil ML, Mant CT, Hodges RS. Role of peptide hydrophobicity in the mechanism of action of alpha-helical antimicrobial peptides. Antimicrob Agents Chemother 2007;51:1398–406. [8] Chen ZS, Tiwari AK. Multidrug resistance proteins (MRPs/ABCCs) in cancer chemotherapy and genetic diseases. FEBS J 2011;278:3226–45. [9] Clinical Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Wayne, PA: CLSI; 2008. Approved Standard M07-A8.

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