Characterization of recombinant plectasin: Solubility, antimicrobial activity and factors that affect its activity

Process Biochemistry 46 (2011) 1050–1055

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Characterization of recombinant plectasin: Solubility, antimicrobial activity and factors that affect its activity Yalin Yang a,b , Da Teng a,b , Jun Zhang a,b , Zigang Tian a,b , Shaoran Wang a,b , Jianhua Wang a,b,∗ a b

Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beijing 100081, PR China Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China

a r t i c l e

i n f o

Article history: Received 7 October 2010 Received in revised form 4 January 2011 Accepted 17 January 2011 Keywords: Recombinant Plectasin Solubility Antimicrobial activity Disulfide bonds Cations

a b s t r a c t Plectasin is the first known fungal defensin with potent activity against Gram-positive bacteria. To evaluate the potential therapeutic application of plectasin, we produced plectasin and investigated its solubility, activity and factors that affect its antimicrobial activity. Recombinant plectasin was produced from Escherichia coli in high yield by integration of fusion expression and on-column cleavage. Including 0.5 M arginine significantly increased the solubility of plectasin in acetic acid buffer with 10% glycerol from 89 ␮g/ml to 408 ␮g/ml. Plectasin was soluble at 846 ␮g/ml in Tris–glycerol–EDTA buffer. Plectasin was active against Gram-positive bacteria Streptococcus pneumoniae and Staphylococcus aureus with minimum inhibitory concentrations of 2 and 0.5 ␮g/ml. Much lower or no activity was observed toward Gram-negative bacteria and fungi. Plectasin (128 ␮g/ml) did not exhibit hemolytic activity toward rabbit erythrocytes. The activity of plectasin toward S. aureus was decreased by reduction with dithiothreitol, indicating that the disulfide-bond is essential for maximal activity. Plectasin was bactericidal under physiological concentrations of mono- and divalent cations. This activity was markedly attenuated by divalent cations in a concentration-dependent manner, however, with complete inhibition occurring at Ca2+ concentrations greater than 25 mM. These results suggested that the presence of the disulfide-bond and the absence of divalent cations play key roles in the antimicrobial activity of plectasin. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Plectasin, the first defensin-like peptide isolated from a saprophytic fungus, contains an ␣–␤ motif consisting of an ␣-helix and two antiparallel ␤-strands and is stabilized by three disulfide bonds (Cys4–Cys30, Cys15–Cys37 and Cys19–Cys39) [1]. It is especially active against Streptococcus pneumoniae, including strains resistant to conventional antibiotics, and was also bactericidal at physiological ionic strength [1]. Mice experimentally infected with peritonitis and pneumonia by S. pneumoniae can be treated with plectasin with results similar to vancomycin and penicillin [1]. Plectasin was not cytotoxic to A549 cells, normal human bronchial epithelial cells or lung fibroblasts and did not induce transcription and production of IL-8 in A549 cells; therefore, it can be considered relatively safe [2]. These results suggested that fungal plectasin could be fur-

∗ Corresponding author at: Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie St., Haidian District, Beijing 100081, PR China. Tel.: +86 10 82106079/82106081; fax: +86 10 82106079. E-mail addresses: [email protected], [email protected] (J. Wang). 1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.01.018

ther emphasized as a potential alternative antibiotic for clinical applications. Plectasin was poorly soluble at neutral pH [1]. Small molecules such as glycerol [3], arginine (Arg) [4], EDTA [5] and DMSO [6] were reported to effectively suppress aggregation and facilitate protein folding. We hypothesized that these small molecules might be used to improve the solubility of plectasin. The conserved disulfide bonds of defensins are thought to play an important role in the structural stabilization [7,8] and biological functions [9–11] of this class of peptides. The functions of the disulfide bonds in plectasin, however, are not well understood. In vitro studies revealed that the microbicidal potencies of various defensins were affected by cations, especially when they were evaluated in the presence of physiologic saline [10–15]. Plectasin showed a bactericidal effect at physiological ionic strength [1], but the exact effect of cations on the activity of plectasin toward Staphylococcus aureus has not been reported. We produced recombinant plectasin in an Escherichia coli expression system and investigated the effects of small molecules on the solubility of the peptide. We also characterized the antimicrobial spectrum and hemolytic activity of recombinant plectasin and studied the effects of disulfide bridges and ions on its antibacterial activity.

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2.4. Effect of small molecules on plectasin solubility

Fig. 1. The codon-optimized nucleotide sequence and corresponding amino acid sequence of recombinant plectasin. The amino acid sequence is indicated by the one-letter code written below the second nucleotide of each codon. BamHI and XhoI restriction sites and the factor Xa cleavage site are indicated by arrows. The amino acid sequence of mature plectasin is underlined. The stop codon is marked with an asterisk.

2. Methods and materials 2.1. Strains and plasmids E. coli DH5␣ (Invitrogen, Beijing, China) was used for propagation of pET-32a(+) (Novagen, Beijing, China) and pETplectasin plasmids; E. coli BL21(DE3) (Novagen) was used as the expression host. The target strains for the antimicrobial activity assay included E. coli CVCC 195(K88), Pseudomonas aeruginosa CVCC 2087, S. aureus ATCC 25923 and S. pneumoniae CVCC 2350 from China Institute of Veterinary Drug Control (Beijing) and Candida albicans CGMCC 2.2411 from China General Microbiological Culture Collection Center (Beijing).

2.2. Recombinant expression and purification of plectasin The optimized plectasin gene was generated by the ‘Reverse Translate Tool’ (http://www.bioinformatics.org/sms2/rev trans.html) according to the codon usage of E. coli (http://www.kazusa.or.jp/codon/). A DNA sequence containing BamHI recognition sites, a protease factor Xa cleavage site, the mature plectasin-encoding gene, a stop codon and XhoI recognition sites was synthesized by Sangon Biotech (Shanghai, China) (Fig. 1). Following digestion with BamHI and XhoI (NEB, Beijing, China), the DNA sequence was ligated into pET-32a(+) to construct the Trx–plectasin-encoding vector pETplectasin. The pETplectasin construct was transferred to E. coli DH5␣, cells were grown on LB agar plates containing 100 ␮g/ml of ampicillin and correctly ligated and transformed plasmids were identified by DNA sequencing. Recombinant E. coli BL21(DE3) was grown at 37 ◦ C to an OD600 of 1.0 in TB medium containing 100 ␮g/ml of ampicillin. Expression was then induced by addition of 0.4 mM isopropyl-1-thio-␤-d-galactopyranoside (IPTG), after which the cultures were incubated at 30 ◦ C for 0, 1, 2, 4, 6 and 16 h. Culture supernatants were harvested by centrifugation (12,000 rpm, 5 min and 4 ◦ C) and analyzed by 12% SDS-PAGE. Cells from the culture that was induced for 4 h were lysed in Bugbuster plus Benzonase (Novagen, 50 mM Tris–Cl, pH 8.0, room temperature, 30 min). The cell lysate was centrifuged (12,000 rpm, 30 min, 4 ◦ C), the supernatant was aspirated, and the pellet was resuspended in 1× lysis mixture. Both soluble and insoluble fractions were analyzed by 12% SDS-PAGE. One gram, wet weight, of cells was resuspended in 5 ml of binding buffer (50 mM NaH2 PO4 , 300 mM NaCl, 10 mM imidazole, pH 8.0) and sonicated to disrupt the cell membranes (150 W, 0 ◦ C, 6 × 5 min of a 30% duty cycle). Cell debris was removed by centrifugation (12,000 rpm, 20 min, 4 ◦ C) and the supernatant was purified with Ni2+ –NTA His·Bind resin pre-equilibrated with 4 column volumes of binding buffer. After washing with binding buffer to baseline absorbance, the fusion protein was eluted at a rate of 1 ml/min with 4 column volumes of elution buffer containing 20, 50, 80, 200, 300 and 500 mM imidazole, respectively. The products were monitored by 12% SDS-PAGE. Protein bands were quantitatively analyzed using Quantity One software (Version 4.6.2, Bio-Rad, USA).

2.3. On-column cleavage of Trx–plectasin and purification of plectasin Supernatants fractions from induced recombinant cells lysate were loaded onto pre-equilibrated Ni2+ –NTA resin and washed with 4 column volumes of elution buffer containing 20 mM imidazole. Following equilibration with factor Xa buffer, the cleavage reaction was run by incubating the column with one column volume of a cleavage buffer containing 20 U factor Xa/ml column volume at 21 ◦ C for 0, 6, 12, 24, 48 and 96 h, respectively. The target peptide and His-tagged fusion protein fractions were eluted with 4 column volumes of cleavage buffer and elution buffer containing 200 mM imidazole successively. The products from 0, 6, 12, 24, 48 and 96 h cleavage were monitored by Tricine–SDS-PAGE. The identity of the target peptide was confirmed by MALDI-TOF at the Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences [16].

Purified plectasin was dialyzed against deionized water in 1000 Da MWCO dialysis tubing, then lyophilized and stored at −20 ◦ C until the activity assay. The effect of four small molecules (10% glycerol, 0.5 M L-Arg, 20% DMSO and 10 mM DTT) on the solubility of plectasin in two solvents was observed. Desalted plectasin was dissolved at a concentration of 1 mg/ml in 0.01% HAc buffer or TGE buffer (50 mM Tris, 0.5 mM EDTA, 50 mM NaCl, 10% glycerol, pH 7.9) containing one to three of the four test compounds. After incubation at room temperature for 24 h with gentle shaking, the supernatant was harvested by centrifugation (15,000 × g, 10 min) and analyzed by Tricine–SDS-PAGE without ␤-mercaptoethanol. Protein bands were quantitatively analyzed using Quantity One software (Version 4.6.2, Bio-Rad, USA). 2.5. Antimicrobial and hemolytic assays The target peptide fraction from the on-column cleavage was further purified on a Superdex Peptide HR 10/30 column (GE Healthcare Bio-Sciences) and eluted with 0.015 M NH4 HCO3 (pH 4.7) at a rate of 0.5 ml/min. The active fractions were lyophilized. The MIC assays were performed using the microdilution broth method [17]. The test organisms were grown to mid-logarithmic phase in Mueller–Hinton broth for Gram-positive bacteria, LB broth for Gram-negative bacteria and YPD broth for yeast, then diluted to 1 × 105 colony forming units (CFU)/ml using corresponding broth. The MH broth was supplemented with 5% defibrinated sheep blood for S. pneumoniae. Aliquots of 100 ␮l of cells (1–5 × 105 CFU/ml) were incubated with twofold peptide dilutions at 35 ◦ C in ambient air for 16–20 h for E. coli and P. aeruginosa or 24 h for S. aureus and S. pneumoniae, or at 28 ◦ C for 24 h for C. albicans. The concentration of the peptide needed for 100% inhibition was designated as the MIC. Each experiment was repeated three times. The hemolytic activity of plectasin was assayed according to a previously published method [18]. Aliquots containing 0.625, 1.25, 2.5, 5, 10, 20, 40, 80, 160, 320, 640 and 1280 ␮g/ml of plectasin in 10 ␮l total volume were added to 90 ␮l of a 2.5% (v/v) suspension of rabbit red blood cells in sterile phosphate-buffered (PBS). After incubation for 30 min at 37 ◦ C, the samples were centrifuged at 10,000 × g for 3 min, and the absorbance of the supernatant was measured at 540 nm. The degree of hemolysis caused by 1% Triton X-100 and PBS buffer was used as the reference value for 100 and 0% lysis, respectively. 2.6. Oxidation and reduction of plectasin Purified plectasin was reduced with 10 mM dithiothreitol (DTT) at 37 ◦ C for 1, 4 and 20 h. Oxidation of plectasin was carried out in aqueous DMSO (20%) for 24 h at room temperature. Both reduced and oxidized plectasins were checked by Tricine–SDS-PAGE without ␤-mercaptoethanol and the inhibition zone method [16]. 2.7. Effect of mono- and divalent cations on the anti-S. aureus activity of plectasin To determine the effect of cations on the activity of plectasin toward S. aureus, samples consisting of 16 ␮g/ml of plectasin, 1 × 105 CFU/ml of S. aureus and 4 cation gradients (0–300 mM NaCl or KCl and 0–50 mM CaCl2 or MgCl2 ) in 100 ␮l total volume of 1 mM phosphate-buffered saline (pH 7.4) were incubated for 3 h at 37 ◦ C, followed by incubation in MH broth for 24 h at 37 ◦ C. The percentage of bacteria that were killed was determined from absorbance measurements at 595 nm.

3. Results 3.1. Expression and purification of Trx–plectasin fusion protein The Trx–plectasin fusion protein appeared as a prominent band at approximately 23 kDa, near its theoretical mass of 22,566.4 Da (Fig. 2A). The yield of fusion protein reached a maximum of 53.5% of the total proteins after a 4-h induction period (Fig. 2A) and 58.5% of it was in a soluble form (Fig. 2B). The fusion protein fraction was purified from the bacteria cell lysates with Ni2+ –NTA His·Bind resin and eluted with 80 mM imidazole, yielding 95.1 mg/l of culture of Trx–plectasin with 94.9% purity (Fig. 2C). 3.2. On-column cleavage of the Trx–plectasin fusion protein with factor Xa Factor Xa, which cleaves the tetrapeptide Ile-Glu-Gly-Arg, was chosen to remove Trx tags from the purified Trx–plectasin. To increase the efficiency of the cleavage and the yield of the purified recombinant peptide, on-column cleavage was used to prepare plectasin. The amount of Trx–plectasin decreased from 90.5% to

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Fig. 2. Expression of recombinant Trx–plectasin fusion protein in E. coli. (A) SDS-PAGE of fusion proteins expressed in E. coli BL21(DE3). Lane 1: total protein from E. coli BL21(DE3); Lanes 2 and 3: total protein from E. coli BL21(DE3) containing pET-32a(+) after induction with IPTG for 0 and 4 h. Lanes 4–9: total protein from E. coli BL21(DE3) containing pETplectasin after induction with IPTG for 0, 1, 2, 4, 6 and 16 h. (B) SDS-PAGE of the insoluble (I), soluble (S) and total cell lysate (T) fractions of Trx–plectasin. (C) SDS-PAGE of purified Trx–plectasin fusion protein. Lanes 1 and 2: insoluble and soluble protein fractions; Lane 3: penetrable apex; Lanes 4–9: elution of purified Trx–plectasin from Ni–NTA His·Bind resin with buffer containing 20, 50, 80, 200, 300 and 500 mM imidazole. Lane M: protein molecular mass standards.

74% during the first 12 h, then to 51% at 96 h, while the amount of carrier protein Trx and cleaved plectasin increased correspondingly (Fig. 3A). After 12 h of on-column cleavage with factor Xa, one small band was observed at the target molecular weight in the Tricine–SDS-PAGE gel (Fig. 3B), and MALDI-TOF analysis revealed one predominant peak from 4399.12 to 4407.12 Da in the target peptide fractions (Fig. 3C). These results suggested that the recombinant plectasin existed as a mixture of states, from the completely oxidized state (calculated molecular mass: 4401.9 Da) with three disulfide bonds, to intermediate states with one or two disulfide bonds, to the completely reduced state (calculated molecular mass:

4407.9 Da) containing six free thiol groups. About 1.75 mg of recombinant plectasin was obtained from 1 l of culture medium. 3.3. Effect of small molecules on solubility of plectasin Plectasin was poorly soluble (89 ␮g/ml) in 0.01% HAc buffer (Fig. 4, Lane 1). Adding 10% glycerol did not improve the solubility of plectasin (Fig. 4, Lane 2). Following the addition of 0.5 M Arg to HAc buffer containing 10% glycerol, the amount of soluble plectasin increased from 89 ␮g/ml to 408 ␮g/ml because the arginine prevented aggregation, resulting in increased native state forma-

Fig. 3. Purification of recombinant plectasin from on-column cleavage with factor Xa. (A) Tricine–SDS-PAGE of the His-tag moiety from Trx–plectasin. Lanes 1–6: elution of the His-tag moiety with elution buffer containing 200 mM imidazole following 0, 6, 12, 24, 48 and 96 h incubation with factor Xa. (B) Tricine–SDS-PAGE of the target peptide moiety from Trx–plectasin. Lanes 1–6: elution of the target moiety with factor Xa buffer following 0, 6, 12, 24, 48 and 96 h incubation with factor Xa. Lane M: protein molecular mass standards. (C) MALDI-TOF mass spectrum of purified recombinant plectasin. Plectasin appeared as two peaks with molecular masses of 4399.12 Da and 4407.12 Da.

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aureus. Plectasin was also active against E. coli CVCC 195(K88) and P. aeruginosa CVCC 2087 at a higher concentration (128 ␮g/ml). C. albicans CGMCC 2.2411 was resistant to recombinant plectasin. Plectasin did not cause hemolysis in rabbit erythrocytes at the concentrations up to 128 ␮g/ml at which it was effective against Gram-negative bacteria (data not shown). This result suggests that plectasin does not impair the integrity of eukaryotic membranes. 3.5. Effect of oxidation and reduction on the anti-S. aureus activity of plectasin

Fig. 4. Effect of small molecules on the solubility of plectasin. Equal amounts of desalted plectasin (1 mg/ml) were dissolved in two solvents, 0.01% HAc buffer (Lanes 1–5) and TGE buffer (Lanes 6–9), with small molecules (10% glycerol, 0.5 M Arg, 20% DMSO and 10 mM DTT) added as indicated. After incubation at room temperature for 24 h with gentle shaking, the supernatants were harvested by centrifugation (15,000 × g, 10 min) and the soluble plectasin content was analyzed by Tricine–SDSPAGE without ␤-mercaptoethanol.

tion from the unfolded or intermediate state. The addition of DMSO produced no effect on the solubility of plectasin (Fig. 4, Lane 3). The highest solubility, 846 ␮g/ml, was obtained in TGE buffer (Fig. 4, Lane 6) and was 9.5 times higher than the solubility in HAc buffer. In TGE buffer, the glycerol stabilizes the plectasin and prevents its aggregation and the EDTA prevents metal-catalyzed air oxidation of cysteines, as previously described [5,19]. However, the solubility of plectasin in TGE buffer decreased about 52–53% upon the addition of Arg or Arg + DMSO (Fig. 4, Lanes 7 and 8). The plectasin band disappeared (Fig. 4, Lanes 5 and 9) when DTT was added into HAc and TGE buffer because it was reduced. 3.4. Antimicrobial and hemolytic assays The antimicrobial activity of recombinant plectasin toward Gram-positive and Gram-negative bacteria and yeast was determined. As shown in Table 1, the MIC (␮g/ml) of plectasin toward S. aureus was double that of penicillin and vancomycin, and the MIC (␮g/ml) of plectasin toward S. pneumoniae was equal to that of vancomycin and eight times lower than that of penicillin. Plectasin was clearly an effective inhibitor of S. pneumoniae and S.

To determine the function of the disulfide bridges of plectasin, the soluble forms of plectasin were oxidized by DMSO or reduced by DTT. The inhibition zone diameters of oxidized (d in Fig. 5A) and reduced plectasin (f in Fig. 5A) were 23 and 9.5 mm, respectively. This result suggests that the disulfide-bond-constrained ␤-sheet tertiary structure is required for maximal antimicrobial activity of plectasin. Gel analysis showed that plectasin that had been reduced for 1 h with DTT traveled faster than the oxidized form (Lanes 1 and 3 in Fig. 5B). Interestingly, some higher molecular weight bands were observed after 4 h of reduction with DTT (Lanes 4 and 5 in Fig. 5B). The higher molecular weight bands may indicate oligomerization of reduced plectasin via intramolecular disulfide bond formation. 3.6. Effects of mono- and divalent cations on the anti-S. aureus activity of plectasin Monovalent cations had no effect on the anti-S. aureus activity of plectasin. Plectasin retained nearly 100% effectiveness in the presence of concentrations of Na+ or K+ up to 150 mM and 97.7% effectiveness even at 300 mM of Na+ or K+ , a value double the normal physiologic concentration (Fig. 6A). Divalent cations inhibited the anti-S. aureus activity of plectasin in a concentration-dependent manner. A limited effect was observed under physiologic concentrations of divalent cations. Plectasin retained 100% and 92.4% of its anti-S. aureus activity at Mg2+ or Ca2+ concentrations of 1.56 mM (Fig. 6B). As the concentration of divalent cations increased, however, the activity of plectasin decreased significantly (Fig. 6B). The anti-S. aureus activity of plectasin was more sensitive to calcium ions than magnesium ions. Plectasin killed 64% and 7.4% of S. aureus in the presence of 6.25 and 12.5 mM Ca2+ , whereas it killed 100% and 90% of S. aureus at the same concentrations of Mg2+ (Fig. 6B). These results demonstrate that divalent cations, especially Ca2+ , play a more important role in the antimicrobial activity of plectasin than monovalent cations.

Table 1 MIC of recombinant plectasin and antibiotics against bacteria and fungi ‘–’ not tested. Strains

Gram-negative E. coli CVCC 195(K88) P. aeruginosa CVCC 2087 Gram-positive S. aureus ATCC 25923

S. pneumoniae CVCC 2350 (C55962) Yeast C. albicans CGMCC 2.2411

Minimal inhibitory concentration (MIC) Plectasin

Kanamycin

Penicillin

Vancomycin

Amphotericin

128 ␮g/ml or 29.04 ␮mol 128 ␮g/ml or 29.04 ␮mol

2 ␮g/ml or 3.43 ␮mol 16 ␮g/ml or 27.46 ␮mol

– –

– –

– –

0.5 ␮g/ml or 0.11 ␮mol

–

0.25 ␮g/ml or 0.70 ␮mol

–

2 ␮g/ml or 0.45 ␮mol

–

16 ␮g/ml or 44.9 ␮mol

0.25 ␮g/ml or 0.17 ␮mol 2 ␮g/ml or 1.35 ␮mol

>128 ␮g/ml or 29.04 ␮mol

–

–

–

32 ␮g/ml or 34.63 ␮mol

–

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Fig. 5. Oxidation and reduction of plectasin. (A) The effect of oxidation and reduction on the anti-S. aureus activity of plectasin. (a) ampicillin (100 ␮g/ml, positive control); (b) plectasin (0.5 mg/ml in TGE buffer); (c) TGE buffer (negative control); (d) plectasin treated with 20% DMSO; (e) TGE buffer containing 20% DMSO; (f) plectasin treated with 10 mM DTT; (g) TGE buffer containing 10 mM DTT; the plate was loaded with 40 ␮l each of (b)–(g). (B) Tricine–SDS-PAGE analysis of plectasin under oxidizing and reducing conditions. Lane 1: plectasin treated with 20% DMSO; Lane 2: plectasin; Lanes 3–5: plectasin incubated with 10 mM DTT at 37 ◦ C for 1, 4 and 20 h; Lane M: protein molecular mass standards.

4. Discussion Defensins from vertebrate [20–23], plant [24,25] and invertebrate [26] sources have been successfully expressed as fusion proteins in E. coli. An important challenge in fusion expression is the release of a tag-free target peptide. Factor Xa can precisely cut the recognition site without leaving additional residues and has been

Fig. 6. Effect of mono- and divalent cations on the anti-S. aureus activity of plectasin.

widely utilized to yield native peptides [16,27–30]. The ‘on-column cleavage’ approach without prior precipitation avoids the loss of fusion proteins from elution and dialysis that occurs in the conventional ‘protease digestion after purification’ method. This method has been successfully used for the purification of antimicrobial peptides [16]. In this work, the cysteine-rich peptide plectasin was expressed in E. coli and purified by on-column cleavage. In the SDS-PAGE analysis, a smaller protein of about 21 kDa was observed exclusively in the soluble fraction (Lane S of Fig. 2B) and purified by Ni–NTA affinity chromatography (Fig. 2C). However, this 21 kDa band could not be detected in the following Tricine–SDSPAGE (Fig. 3A). The smaller protein in the soluble fraction may have resulted from normal folding or misfolding and even other unknown folding way of Trx–plectasin caused by its uniquely disulfide bonds, its final conclusion needs to be verified in next work. Poor aqueous solubility of proteins is a common problem in the development of drugs [31]. Small molecules such as glycerol [3] and arginine [4] are widely used to prevent misfolding and aggregation of proteins. In this study, the addition of arginine improved the solubility of plectasin in acetic acid buffer with 10% glycerol. The activity spectrum of the recombinant plectasin was evaluated against a panel of bacteria and fungi. Plectasin was active in vitro against Gram-positive bacteria but exhibited limited or no activity against Gram-negative bacteria and fungi. These results are consistent with previous reports [1,2]. Mygind et al. reported that plectasin was neither cytotoxic to murine fibroblasts and human epidermal keratinocytes nor hemolytic to human erythrocytes [1]. Similarly, hemolysis of rabbit erythrocytes by plectasin was not observed in this study. The disulfide bonds of defensins play important roles in stabilizing their structures. The disulfide arrangement of Crp4 was shown to protect the peptide from degradation by matrix metalloproteinase-7 [7]. The disulfide bridges of human defensin contributed to the stability of the structure of the molecule and to maintenance of the activity [8]. Some results have suggested that the disulfide bonds of defensin play an important role in its antimicrobial activity. Disulfide bridges were necessary for the activity of Tenecin 1 [9]. Campopiano et al. concluded that the activity and stability of a mixture of Defr1 dimeric isoforms was enhanced by the presence of an intermolecular disulfide bond [10]. Some research, however, has suggested that the antimicrobial activity of defensins is independent of disulfide bridges. Wu et al. recently demonstrated

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that the antimicrobial activity of human ␤-defensin 3 (HBD3) was not affected by its disulfide bridges, although the chemotactic properties of this peptide were dependent on disulfide bond formation [11]. Mandal et al. reported that a rigid ␤-sheet structure or the presence of three disulfide bridges did not appear to be stringent requirements for antibacterial activity in ␤-defensins [32]. Our results from the reduction and oxidization of plectasin indicated that the disulfide-bond-constrained ␤-sheet tertiary structure is required for maximal antimicrobial activity. Further work focusing on these differences remains to be done. Many defensins, such as mammalian defensins [10–14] and avian defensin [15], demonstrated salt-sensitive microbicidal activity. Although inhibition effects have been observed for both mono- and divalent cations, these inhibition effects depended on the total ionic strength of the medium [33]. A higher ionic strength may hamper the electrostatic interaction between the positively charged defensin and the negatively charged microbial membrane surface [34,35] King penguin spheniscin 2 (Sphe-2), however, retained antimicrobial activity at high cation concentrations [36]. This discrepancy was assumed to result from differences in the net positive charge of the peptide. Sphe-2 has a higher net positive charge (+11) than other defensins, which may reduce the ability of high cation concentrations to interfere with microbicidal activity [36]. Although plectasin had a low net positive charge (+1 to +3), it still exhibited bactericidal activity at the relatively high ionic strength of physiological saline [1]. In this study, the bactericidal activity of plectasin was inhibited by divalent cations such as Ca2+ and Mg2+ but not by monovalent cations. Our results suggest that the antimicrobial activity of plectasin might involve a specific interaction of plectasin with the surface of the target microorganism and support a previous report that plectasin directly bound the bacterial cell-wall precursor Lipid II [37]. Further work remains to explore the detailed relationship between plectasin, Lipid II and divalent cations. Our conclusions about the contribution of disulfide bonds and ions to the antibacterial activity of recombinant plectasin will be useful for further research into the antimicrobial mechanism of plectasin and for potential drug development.

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