Brevinin-1BYa: a naturally occurring peptide from frog skin with broad-spectrum antibacterial and antifungal properties

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Brevinin-1BYa: a naturally occurring peptide from frog skin with broad-spectrum antibacterial and antifungal properties

Tibor P´al

^a

, Bency Abraham

^b

, ´ Agnes Sonnevend

^a

, Pauline Jumaa

^a

, J. Michael Conlon

^b,∗

aDepartment of Medical Microbiology, Faculty of Medicine and Health Sciences, United Arab Emirates University, 17666 Al-Ain, United Arab Emirates

bDepartment of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, 17666 Al-Ain, United Arab Emirates Received 24 November 2005; accepted 19 January 2006

Abstract

Brevinin-1BYa (FLPILASLAAKFGPKLFCLVTKKC) is a cationic␣-helical peptide containing an intramolecular disulphide bridge that is present in skin secretions of the foothill yellow-legged frogRana boylii. A synthetic replicate of the peptide showed growth inhibitory activity against a range of reference strains of Gram-positive and Gram-negative bacteria, against clinical isolates of methicillin-resistant Staphylococcus aureus(MRSA) (minimum inhibitory concentration (MIC) = 2.5␮M), and against reference strains and clinical isolates of the opportunistic yeast pathogensCandida albicans,Candida tropicalis,Candida kruseiandCandida parapsilosis(MIC≤10␮M). However, the therapeutic potential of the peptide, especially for systemic applications, is restricted by its high haemolytic activity against human erythrocytes (LD50= 10␮M). Replacement of the cysteine residues in brevinin-1BYa by serine produced an acyclic analogue with eight-fold reduced haemolytic activity that retained high potency against Gram-positive bacteria, including strains of MRSA (MIC = 5␮M), however activities against Gram-negative bacteria and yeast species were reduced. It is suggested that brevinin-1BYa represents a candidate for drug development, particularly for topical applications against antibiotic-resistant microorganisms.

Keywords: Antibacterial and antifungal peptide; Brevinin-1; Frog skin

1. Introduction

The emergence, particularly during the past 10–15 years, of strains of pathogenic bacteria and fungi that are resistant to commonly used antibiotics poses a potentially serious threat to public health[1]. New types of antimicrobial agents with activity against these microorganisms are required that also possess appropriate pharmacokinetic and toxicological pro- files. Peptides with antimicrobial activity play an important role in the innate immunity that pre-dates adaptive immunity and constitutes the first-line of defence against invading pathogens for a wide range of vertebrate and invertebrate species[2,3]. Compounds based upon these naturally occurring anti-infective peptides are being increasingly considered

∗Corresponding author. Tel.: +971 3 713 7484; fax: +971 3 767 2033.

E-mail address:[email protected](J.M. Conlon).

as potential therapeutic agents [4,5]. The mechanism by which peptides produce cell death does not involve binding to specific receptors on the cell membrane, but rather a non-specific interaction with the membrane (phospho)lipids [6]. Consequently, development of resistance to antimicrobial peptides takes place at rates that are orders of magnitude lower than those observed for conventional antibiotics [7].

The major obstacles to their development as useful drugs, particularly if they are to be administered systemically, are their cytolytic activities against mammalian cells and their short half-lives in the circulation. However, peptides applied to infected skin or skin lesions in the form of sprays or ointments can penetrate into the stratum corneum to kill microorganisms, therefore future therapeutic applications are more likely to involve topical rather than systemic administration[8,9].

Frogs of the genusRana(Ranidae), a group of ca. 250 extant species distributed worldwide, have proved to be a

doi:10.1016/j.ijantimicag.2006.01.010

ANTAGE-2092; No. of Pages 5

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particularly rich source of peptides with antibacterial and antifungal activities [10]. These peptides are synthesised in granular glands in the skin and are released into skin secretions, often at very high concentration, in response to environmental stress that can be mimicked in the laboratory by mild electrical stimulation or injection of noradrenalin.

There are no conserved amino acid sequences that are associated with antimicrobial activity, but theRanaskin peptides are almost invariably cationic, relatively hydrophobic and have the propensity to form an amphipathic ␣-helix in a membrane-mimetic solvent[10,11].

The 24 amino acid peptide brevinin-1BYa represents a candidate for drug development. The peptide was first isolated from noradrenalin-stimulated skin secretions from the North American foothill yellow-legged frog Rana boylii and preliminary data indicated potent growth inhibitory activity against reference strains of Candida albicans and Staphylococcus aureus(minimum inhibitory concentration (MIC)≤5␮M) [12]. The present study investigates the growth inhibitory activity of a synthetic replicate of this peptide against a range of reference strains of Gram- positive and Gram-negative bacteria and against fourCan- didaspecies. In addition, activities against clinical isolates of methicillin-resistantS. aureus(MRSA) andCandidaspp.

have been determined. As brevinin-1BYa displays strong haemolytic activity against human erythrocytes, an analogue with reduced haemolytic activity was synthesised and its antimicrobial properties compared with the naturally occurring peptide.

2. Materials and methods 2.1. Bacteria

Reference strains were purchased from the American Type Culture Collection (ATCC, Rockville, MD), from the National Collection of Type Cultures (London, UK) and from the Hungarian National Type Culture Collection (Budapest, Hungary) and have been described previously [13]. Six strains of MRSA were isolated from wounds of patients admitted to Tawam Hospital (Al-Ain, United Arab Emi- rates). Their oxacillin MIC values, as determined by Etest (AB Biodisk, Solna, Sweden), varied between 16 mg/L and

>256 mg/L. ThemecAgene, encoding the penicillin-binding protein PBP2a, was shown to be present in all strains by poly- merase chain reaction[14].

2.2. Yeasts

Reference strains were purchased from the ATCC. Clinical isolates ofC. albicans,Candida parapsilosis,Candida trop- icalisandCandida kruseiwere obtained from patients with episodes of candidaemia from Tawam Hospital. All strains tested were susceptible to amphotericin B. The C. krusei strains were resistant to antifungal azoles.

2.3. Peptides

Brevinin-1BYa (FLPILASLAAKFGPKLFCLVTKKC) and [Ser¹⁸,Ser²⁴]brevinin-1BYa (FLPILASLAAKFGPKLF- SLVTKKS) were supplied in crude form by GL Biochem (Shanghai) Ltd. (Shanghai, China) and purified to near homogeneity by reverse-phase high-performance liquid chromatography on a 2.2 cm×25 cm Vydac 218TP1022 (C- 18) column (Separations Group, Hesperia, CA) equilibrated with acetonitrile/water/trifluoroacetic acid (21.0/78.9/0.1) at a flow rate of 6 mL/min. The concentration of acetonitrile was raised to 56% over 60 min using a linear gradient. The final purity of the synthetic peptides was >95% and their identities were confirmed by electrospray mass spectrometry.

2.4. Antimicrobial assays

MICs of the peptides were determined in duplicate in two independent experiments by standard microdilution methods using 96-well microtitre cell culture plates[15,16]. Serial dilutions of the peptides in Mueller–Hinton broth (50␮L) were mixed with an inoculum (50␮L of 10⁶colony-forming units (CFU)/mL) from a log-phase culture. Bacteria were incubated for 18 h at 37^◦C in a humidified atmosphere of air.

Candidaspp. were tested in RPMI-1640 medium for 48 h at 35^◦C with an inoculum of 5×10⁴CFU/mL. After incubation, the absorbance at 630 nm of each well was determined using a microtitre plate reader. To monitor the validity and reproducibility of the assays, incubations with bacteria were carried out in parallel with increasing concentrations of bac- itracin, and incubations withCandida spp.in parallel with amphotericin B.

2.5. Haemolysis assay

Peptides in the concentration range 1–100␮M were incubated with washed human erythrocytes (2×10⁷cells) from healthy donors 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 of the supernatant at 450 nm was measured. A parallel incubation in the presence of 1% v/v Tween-20 was carried out to determine the absorbance associated with 100% haemolysis. The LD50value was taken as the mean concentration of peptide producing 50% haemolysis in three independent experiments.

3. Results

The MIC values of synthetic replicates of brevinin-1BYa and [Ser¹⁸,Ser²⁴]brevinin-1BYa against reference strains of bacteria and the haemolytic activity (␮M) against human erythrocytes are shown inTable 1. Removal of the intramolecular disulphide bridge of brevinin-1BYa by replacement of the Cys¹⁸ and Cys²⁴ residues with serine residues resulted in an eight-fold reduction in haemolytic activity, but this

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Table 1

Minimum inhibitory concentrations (␮M) of synthetic replicates of brevinin- 1BYa and [Ser¹⁸,Ser²⁴]brevinin-1BYa against reference strains of bacteria and haemolytic activity against human erythrocytes (LD50)

Reference strain Brevinin-

1BYa

[Ser¹⁸, Ser²⁴]Brevinin- 1BYa

Escherichia coliATCC 25922 20 40

Klebsiella pneumoniaeKK3 9904 40 >40

Enterobacter cloacaeHNTCC 53001 10 40

Pseudomonas aeruginosaATCC 27853 10 40 Proteus mirabilisATCC 25933 >40 >40

Staphylococcus aureusNCTC 8325 2.5 5

Staphylococcus epidermidisRP62A 5 5

Enterococcus faecalisATCC 29212 5 20

StreptococcusGroup B HNTCC 80130 10 10

LD50erythrocytes (␮M)^a 10 80

aMean concentration of peptide producing 50% haemolysis in three independent experiments.

substitution also resulted in appreciable decreases in antimicrobial potency, particularly against Gram-negative bacteria (up to four-fold).

The growth inhibitory properties of brevinin-1BYa and [Ser¹⁸,Ser²⁴]brevinin-1BYa against six strains of multidrug- resistant MRSA and the resistance of the strains to commonly used antibiotics are shown inTable 2. The MIC values against the clinical isolates were the same as against the ATCC reference strain, and Cys→Ser substitution resulted in only a two- fold reduction in potency. Brevinin-1BYa showed relatively high potency (MIC≤10␮M) both against reference strains and clinical isolates of the opportunistic yeast pathogens C. albicans, C. tropicalis, C. krusei and C. parapsilosis (Table 3). The potency of the serine-substituted analogue against these microorganisms was appreciably less that that of the cyclic peptide and the compound was inactive against C. kruseiat concentrations up to 40␮M.

4. Discussion

Brevinin-1 was first isolated from an extract of the skin of the Japanese pond frog Rana brevipoda porsa[17] but subsequent work has shown that orthologous peptides are

Table 3

Minimum inhibitory concentrations (␮M) of synthetic replicates of brevinin- 1BYa and [Ser¹⁸,Ser²⁴]brevinin-1BYa against reference strains and clinical isolates ofCandidaspp.

Strain Brevinin-

1BYa

[Ser¹⁸,Ser²⁴]Brevinin- 1BYa

C. albicansATCC 90028 5 40

C. albicansV2414 5 40

C. albicansV3577 5 40

C. parapsilosisATCC 22019 5 40

C. parapsilosis3804 5 40

C. parapsilosisV65A 10 >40

C. tropicalisV1757 5 20

C tropicalisV3117 5 20

C. tropicalisB51a/5 5 10

C. kruseiATCC 6258 5 >40

C. kruseiV5800 5 >40

C. krusei4426 10 >40

synthesised in the skin of a wide range of Eurasian and North American ranid species[10]. Circular dichroism studies have demonstrated that brevinin-1 peptides exist predominantly as a random coil in aqueous solution but adopt an extended␣- helical conformation in a membrane-mimetic environment such as 50% trifluoroethanol, with the strongly conserved Pro¹⁴ residue introducing a stable kink into the molecule [18,19]. Structure–activity studies with brevinin-1 analogues indicate that the N-terminal domain of the peptide (residues 1–13) is the most important region for determining antimicrobial activity [20]. The disulfide bridge is not necessary for high potency but replacement of the cysteine residues by serine causes the conformation of the C-terminal region to become completely disordered[19]. Naturally occurring acyclic brevinin-1 orthologs that show strong antimicrobial activity have been identified in the skin of certain ranid frogs [21].

The present study has shown that a synthetic replicate of brevinin-1BYa shows growth inhibitory activity against reference strains of Gram-negative and Gram-positive bacteria and several yeast species. Brevinin-1BYa was not active against Proteus mirabilis (MIC > 40␮M) and it is speculated that proteolytic enzymes released by this microorganism rapidly degrade the peptide[22]. Of particular interest, the peptide

Table 2

Minimum inhibitory concentrations (MICs) of brevinin-1BYa and [Ser¹⁸, Ser²⁴]brevinin-1BYa against clinical isolates of methicillin-resistantStaphylococcus aureus(MRSA) with varying degrees of antibiotic resistance

MRSA strain Susceptibility/resistance MIC (␮M)

CLI GEN AMK RIF ATM ERY FUS TEIC LZD SXT CIP LVX TET Brevinin-

1BYa

[Ser¹⁸,Ser²⁴] Brevinin-1BYa

T7/5 S R S S S S S S S R R R R 2.5 5

T7/20 S R S R R R S S S R R S R 2.5 5

T17/13 R R R S R R S S S R R R R 2.5 5

T27/9 S R R S R R R S S S R R R 2.5 5

T58/7 S R R S R R R S S S R R R 2.5 5

V4180 S R R R R R S S S R R R R 2.5 5

CLI, clindamycin; GEN, gentamicin; AMK, amikacin; RIF, rifampicin; ATM, aztreonam; ERY, erythromycin; FUS, fusidic acid; TEIC, teicoplanin; LZD, linezolid; SXT, sulphamethoxazole/trimethoprim; CIP, ciprofloxacin; LVX, levofloxacin; TET, tetracycline; R, resistant; S, susceptible.

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has high potency against two groups of clinically important pathogens for which antibiotic resistance has become a serious problem, namely MRSA andCandidaspp. Methicillin resistance first appeared among nosocomial isolates of S.

aureusin 1961[23]and since that time MRSA has emerged to become a major phenotype in hospitals worldwide, with a high rate of mortality[24]. More recently, new strains of MRSA have emerged in the community causing infections in young, otherwise healthy people[25]. In addition to␤-lactam resistance, MRSA strains may exhibit multidrug resistance, including non-susceptibility to quinolones, macrolides and sulphonamides[26]. Similarly, the widespread use of azoles has led to the rapid development of multidrug resistance in C. albicans and other Candida species, which poses a major problem for antifungal therapy [27]. Patients under- going prolonged immunosuppressive therapy for transplants or treatment of malignancy[28]and patients with indwelling devices[29]are particularly at risk for nosocomialCandida infections.

The strong haemolytic activity of brevinin-1BYa against human erythrocytes (LD50= 10␮M) severely limits the potential of the peptide for development into a therapeuti- cally valuable anti-infective drug, particularly for systemic use. The antimicrobial activities of a peptide against bacteria and fungi and the cytolytic activity against mammalian cells are determined by a complex interaction between cationicity, hydrophobicity, ␣-helicity and amphipathicity [6,11,30].

Studies using model␣-helical peptides have demonstrated that an increase in both the degree of ␣-helicity and the degree of amphipathicity promotes haemolytic activity relative to antimicrobial activity [31,32]. It is probable, therefore, that the appreciably (eight-fold) lower haemolytic activity of [Ser¹⁸,Ser²⁴]brevinin-1BYa compared with the native peptide arises from destabilisation of the␣-helix in the C-terminal region of the peptide associated with replacement of the cystine bridge [19]. The acyclic analogue retained high antimicrobial potency against Gram-positive bacteria, including the MRSA strains, but activities against Gram- negative andCandidaspecies were appreciably reduced.

The bacterial cytoplasmic cell membrane is rich in acidic phospholipids, whereas the plasma membrane of mammalian cells such as human erythrocytes contains a much higher proportion of zwitterionic phosphatidylcholine and sphin- gomyelin phospholipids. Therefore, an increase in peptide cationicity should promote interaction with the more nega- tively charged bacterial cell membrane and increase antimicrobial potency without increasing haemolytic activity. This prediction has been confirmed in the authors’ laboratories by the synthesis of more potent analogues of the frog skin antimicrobial peptides, pseudin-2 [13]and kassinatuerin-1 [33], in which selected neutral amino acids were replaced by lysine. It is therefore proposed to adopt this strategy in future studies to develop analogues of [Ser¹⁸, Ser²⁴]brevinin- 1BYa with increased antimicrobial potency by increasing cationicity while retaining the amphipathic␣-helical conformation.

Acknowledgments

This work was supported by an Interdisciplinary Grant (03/12-8-03-01) and a Faculty Support Grant (NP/05/01) from the United Arab Emirates University.

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