Design and Recombination Expression of a Novel Plectasin-Derived Peptide MP1106 and Its Properties Against Staphylococcus Aureus

Appl Microbiol Biotechnol DOI 10.1007/s00253-014-6077-9

BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

Design and recombination expression of a novel plectasin-derived peptide MP1106 and its properties against Staphylococcus aureus

Xintao Cao & Yong Zhang & Ruoyu Mao & Da Teng & Xiumin Wang & Jianhua Wang

Received: 4 August 2014 /Revised: 5 September 2014 /Accepted: 7 September 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract A novel antimicrobial peptide MP1106 was de- hemolytic activity of only 1.16 % at a concentration of signed based on the parental peptide plectasin with four mu- 512 μg/ml and remained stable in human serum at 37 °C for tational sites and a high level of expression in Pichia pastoris 24 h. Furthermore, the activity of rMP1106 was minorly X-33 via the pPICZαA plasmid was achieved. The concen- affected by 10 mM dithiothreitol and 20 % dimethylsulfoxide. tration of total secreted protein in the fermented supernatant Our results indicate that MP1106 can be produced on a large was 2.134 g/l (29 °C), and the concentration of recombinant scale and has potential as a therapeutic drug against S. aureus. MP1106 (rMP1106) reached 1,808 mg/l after a 120-h induction in a 5-l fermentor. The rMP1106 was purified using a Keywords MP1106 . Antimicrobial peptide . Pichia cation-exchange column, and the yield was 831 mg/l with pastoris . Stability . Staphylococcus aureus 94.68 % purity. The sample exhibited a narrow spectrum against some Gram-positive bacteria and strong antimicrobial activity against Staphylococcus aureus at low minimal inhib- Introduction itory concentrations (MICs) of 0.014, 1.8, 0.45, and 0.91 μM to S. aureus strains ATCC 25923, 29213, 6538, and 43300, The overuse of antibiotics has led to the emergence of respectively. Meanwhile, rMP1106 showed potent activity antibiotic-resistant pathogenic bacteria that has become a huge (0.03–1.8 μM) against 20 clinical isolates of methicillin- threat to public health. Many types of Staphylococcus aureus resistant S. aureus (MRSA). In addition, rMP1106 exhibited cause cutaneous and soft tissue infections (Miller and Cho a broad range of thermostability from 20 to 100 °C. The higher 2011). However, treating these infections is difficult because antimicrobial activity of rMP1106 was maintained in neutral of the emergence of multidrug-resistant strains (Lowy 1998). and alkaline environments (pH 6, 8, and 10), and its activity Methicillin was introduced into clinical use in 1960, and the was slightly reduced in acidic environments (pH 2 and 4). The first observations of methicillin-resistant S. aureus (MRSA) rMP1106 was resistant to the digestion of pepsin, snailase, and were reported in 1961 (Woodford and Livermore 2009). In proteinase K and was sensitive to trypsin. It exhibited recent years, infections caused by MRSA were detected in hospitals (hospital-acquired MRSA (HA-MRSA)), communi- Xintao Cao, Yong Zhang, and Ruoyu Mao contributed equally to this ties (community-acquired MRSA (CA-MRSA)), and live- paper. stock (livestock-acquired MRSA (LA-MRSA)) in a range of X. Cao : Y. Zhang : R. Mao : D. Teng : X. Wang : J. Wang animal species (Chambers and Deleo 2009;Kocketal.2011). Key Laboratory of Feed Biotechnology, Ministry of Agriculture, In addition, the CC398 strain of LA-MRSA, originally found Beijing 100081, China in pigs and cattle, was surprisingly detected in humans in 2003 : : : : : * (Graveland et al. 2011; van Cleef et al. 2011). Moreover, X. Cao Y. Zhang R. Mao D. Teng X. Wang J. Wang ( ) β Gene Engineering Laboratory, Feed Research Institute, Chinese MRSA strains are resistant to all members of the -lactam Academy of Agricultural Sciences, 12 Zhongguancun Nandajie St., class of antibiotics, including all penicillins, cephalosporins, Haidian District, Beijing 100081, People’sRepublicofChina and carbapenems, because they can produce penicillin- e-mail: [email protected] binding proteins (PBPs), known as PBP2 or PBP2a, that have β J. Wang low affinity to most -lactams and therefore confer resistance e-mail: [email protected] (Stefani et al. 2012; Woodford and Livermore 2009). Appl Microbiol Biotechnol

Therefore, an urgent need exists for discovering and develop- Peptide design ing new antimicrobial drugs to control widespread MRSA. Antimicrobial peptides (AMPs) are potent pharmaceuticals The main structure of plectasin (α-helix from residues known for their rapid and broad-spectrum antimicrobial prop- 12–20 and β-sheets in residues 27–31 and 36–40) was erties, particularly those against antibiotic-resistant bacteria not changed to maintain or improve its expression level (Brogden and Brogden 2011). Plectasin, the first fungal in P. pastoris. Additionally, the amino acids containing a defensin with therapeutic potential that was especially targeted positive net charge and hydrophobic groups were intro- against Gram-positive bacteria, was identified from the sapro- duced to increase the antimicrobial activity of plectasin. phytic ascomycete Pseudoplectania nigrella (Mygind et al. As a result, S, K, and H residues containing hydroxyls 2005). This compound kills the pathogens by directly binding and a positive net charge were used to replace the polar Lipid II, the essential precursor of the cell wall (Schneider amino acid residues of D and Q at positions 9, 11, and et al. 2010). Additionally, plectasin showed no cytotoxicity to 14. Additionally, I, which contains a larger hydrophobic eukaryotic cells, a low induction of interleukin-8 (IL-8) pro- side chain, was used to replace the V residue in position duction (Hara et al. 2008), and no specific antibody responses 36. The amino acid sequences and properties of the in mice (Brinch et al. 2009). It also can be easily produced by parent peptides and designed peptide were calculated by prokaryotic and eukaryotic expression systems (Mygind et al. bioinformatics programs, including Antimicrobial Peptide 2005;Rothanetal.2013;Yangetal.2011; Zhang et al. 2011). Calculator and Predictor (http://aps.unmc.edu/AP/ The minimal inhibitory concentrations (MICs) of plectasin prediction/prediction_main.php), ProtParam (ExPASy against Streptococcus pneumonia and Staphylococcus Proteomics Server: http://www.expasy.org/tools/ epidermidis were 0.063–8and4–32 μg/ml, respectively. protparam.html), and Pepstats (EMBOSS biology However, the MICs of plectasin to S. aureus were 4– software: http://www.ebi.ac.uk/Tools/emboss/pepinfo/). 128 μg/ml (Hara et al. 2008;Mygindetal.2005), suggesting The mean hydrophobicity and relative hydrophobic plectasin activity needed to be increased to be effective against moment were calculated online using CCS scale (http:// S. aureus. www.bbcm.univ.trieste.it/wtossi/HydroCalc/HydroMCalc. The development of AMPs is still hampered by their tox- html). All of the parameters are summarized in Table 1. icity to mammalian cells and the lack of a cost-effective means The spatial structure of MP1106 was calculated by of commercial-scale production (Brogden 2005; Findlay et al. SWISS-Model (http://swissmodel.expasy.org/workspace// 2010; Brogden and Brogden 2011). A variety of approaches index.php?func=modelling_simple1&userid= have been employed to improve antimicrobial activity and USERID&token=TOKEN). reduce undesirable cytotoxic effects through optimization of natural AMP sequences (Chen et al. 2005; Jiang et al. 2011; Construction of the expression vector pPICZαA-MP1106 Lee et al. 2014). In this study, a variant of plectasin named MP1106 was designed and expressed in Pichia pastoris A 120-bp codon-optimized gene sequence of MP1106 was (named rMP1106). Additionally, the bactericidal activity, he- designed using the Reverse Translate Tool (www.bio- molytic activity, and stability of purified rMP1106 were informatics.org/sms2/rev_trans.html) based on the evaluated. preferential codon usage of P. pastoris (www.kazusa.or.jp/ codon/) (Yang et al. 2011). The DNA fragment contained the XhoI restriction site, a P. pastoris Kex2 protease Materials and methods cleavage site, the codon-optimized MP1106 open reading frame (ORF), two stop codons, and the XbaI restriction site Strains, plasmids, and reagents and was synthesized by Sangon Biotech (Shanghai, China). The DNA fragment and pPICZαA vectors were digested with Escherichia coli DH5α (Invitrogen, Beijing, China), XhoI and XbaI, gel-purified, and ligated together with T4 P. pastoris X-33 (Invitrogen, Beijing, China), and pPICZαA DNA ligase. The recombinant plasmid pPICZαA-MP1106 vectors (Invitrogen, Beijing, China) were used for cloning and was transformed into E. coli DH5α, and positive cells were expression. All of the strains used in antimicrobial assays and selected on zeocin-containing LB plates (25 μg/ml) and were their sources are summarized in Table 2. Vancomycin and screened by colony PCR with the following two pairs of ampicillin were purchased from the China Institute of Veter- primers: inary Drug Control. DNA restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (NEB, Primer 5′ AOX1: 5′-GACTGGTTCCAATTGACAAGC-3′ Beijing, China). The kits for plasmid extraction and DNA Primer 3′ AOX1: 5′-GCAAATGGCATTCTGACATCC-3′ purification were purchased from Tiangen (China). All other F1: 5′-CCGCTCGAGAAGAGAGGTTT-3′ chemical reagents used were of analytical grade. R1: 5′-GCTCTAGATTATTAGTAACAC-3′ Appl Microbiol Biotechnol

Table 1 Sequences and key physicochemical parameters of Plectasin and MP1106

Peptides Sequence Mw pI GRAVYb μHrelc Charge

Plectasin GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 4,401.99 7.77 −0.695 0.82 +2.0 MP1106 GFGCNGPWSEKDMHCHNHCKSIKGYKGGYCAKGGFICKCYa 4,410.12 8.84 −0.622 0.68 +5.5 a The underlined sites of MP1106 was four mutation sites compared with plectasin b The grand average of hydropathicity (GRAVY) of a peptide is the sum of hydropathy values of all the amino acids divided by the numbers of residues in the peptide or protein sequence; a negative value represents a hydrophilic protein c The relative hydrophobic moment (μHrel) is its hydrophobic moment relative to that of a perfectly amphipathic peptide. A value of 0.5 thus indicates that the peptide has about 50 % of the maximum possible amphipathicity

Transformation and selection of positive transformants colony of an rMP1106 transformant was cultured in a shaking flask containing 10 ml of YPD (100 μg/ml zeocin) at 29 °C The pPICZαA-MP1106 vector was linearized by PmeIand and 250 rpm overnight. The overnight culture was inoculated was then transformed into P. pastoris X-33 by electroporation into 200-mL fresh YPD medium and cultivated at 29 °C to an with 1,200 V of charging voltage, 25 μF of capacitance, and OD600 nm of 4.0–6.0 and then transferred into a 5-l fermentor 200 Ω of resistance. The pPICZαA vector was used as a containing 1.8 l basal salt medium (50 g/l NH4H2PO4,20g/l negative control. Positive transformants were selected on K2SO4,15g/lMgSO4·7H2O, 6 g/l KH2PO4, 0.4 g/l CaSO4, YPDS (1 % yeast extract, 2 % peptone, 2 % glucose, 1 M and 1.5 g/l KOH) and 200 ml 45 % (w/v)glucose.The sorbitol, and 2 % agar) plates containing 100 μg/ml zeocin. induction process contained three successive phases. First, Then, the genomic DNA from recombinant yeast was pre- 200 ml of 50 % (w/v) glucose was added to support cell pared using a “boiling-freezing-boiling” method. Positive growth once glucose was exhausted. Second, methanol was transformants were further confirmed by PCR with the spe- supplied from 1 to 6 ml/l/h once glucose was exhausted. cific primers mentioned above. Finally, methanol was supplied to maintain a relative dissolved oxygen (DO) content between 20–40 % under the Expression of rMP1106 in P. pastoris in shake flasks speed of 6 to 8 ml/l/h. The temperature and pH value were adjusted and maintained at 29 °C and 5.5, respectively. The A single colony of positive P. pastoris transformants was fermentation liquid was collected every 12 h to quantify the cultured at 29 °C and 250 rpm in 50-ml shaking flasks con- cell wet weight, and the total protein yield was assayed using a taining 20-ml BMGY medium (1 % yeast extract, 2 % pep- Bradford protein assay kit (Tiangen Biotech, Beijing, China). tone, 1 % glycerol, 1.34 % yeast nitrogen base, 0.004 % The expression of rMP1106 was determined using an inhibition zone assay and Tricine-SDS-PAGE. biotin, 0.1 M PBS, pH 6.0) until the OD600 nm reached approximately 5.0. The cells were harvested by centrifugation at 6,000×g for 2 min at room temperature and were resus- Purification of rMP1106 pended in 50-ml BMMY medium (1 % yeast extract, 2 % peptone, 0.5 % methanol, 1.34 % yeast nitrogen base, 0.004 % The fermentation broth was centrifuged at 5,000 rpm for biotin, 0.1 M PBS, pH 6.0) to an OD600 nm of 1.0; the cells 30 min at 4 °C, and the supernatant was lyophilized. The were then cultured at 29 °C at 250 rpm to induce protein dry powder was dissolved in distilled water and was then α expression. Meanwhile, the P. pastoris strain X-33/pPICZ A applied onto an SP sepharose FF cation-exchange column was used as negative control. Methanol was added every 24 h (GE Healthcare, UK) pre-equilibrated with binding buffer during the 120-h induction period to a final concentration of (20 mM sodium phosphate buffer, pH 6.7). The rMP1106 approximately 0.5 % (v/v). The expression of MP1106 was was eluted from the column with elution buffer (20 mM determined using the inhibition zone assay against S. aureus sodium phosphate buffer, 600 mM NaCl, pH 6.7) at a rate of ATCC 25923 and Tricine-sodium dodecyl sulfate polyacryl- 6 ml/min. The protein elution was monitored by measuring amide gel electrophoresis (Tricine-SDS-PAGE) (Schagger UV absorbance at 215 and 280 nm, and the highest peak 2006). absorbance fractions were pooled, desalted, and freeze-dried for subsequent antimicrobial assays. The purified rMP1106 Expression of rMP1106 in a 5-l fermentor samples were analyzed by Tricine-SDS-PAGE, and the pu- rities of the target protein bands were quantified by using To improve the production of rMP1106, a 5-l fermentor Quantity One software (version 4.6.3, Bio-Rad, USA). The (BIOSTATB plus, Sartorius Stedim Biotech) and the highest purified rMP1106 was also confirmed by matrix-assisted laser expression P. pastoris-rMP1106 strain were used. A single desorption/ionization-time of flight mass spectrometry Appl Microbiol Biotechnol

(MALDI-TOF MS) at the Laboratory of Proteomics, Institute 105 CFU/ml. A 90-μl cell suspension and 10 μl of serial of Biophysics at the Chinese Academy of Sciences. concentration gradient solutions of rMP1106 were added to the wells of 96-well cell culture plates. The plates were incu- Antimicrobial activity assay bated at 37 °C for 18–24 h. The antimicrobial activity of vancomycin and ampicillin were also tested as positive con- 1. Antimicrobial activity of purified rMP1106 was analyzed trols. Experiments were performed in triplicate. The MIC was by agar well diffusion (Tian et al. 2009a). The S. aureus determined to be the lowest concentration at which observable

strain ATCC 25923 was cultivated to an OD600 nm of 0.4 growth was inhibited. Minimum bactericidal concentration in Muller-Hinton broth medium (5 g/l beef infusion (MBC) was determined by plating 10-μl samples from wells solids, 17.5 g/l casein hydrolysate, 1.5 g/l starch, pH with no visible growth onto MHA plates and is the lowest 7.4) at 37 °C. A total of 1 % of the cell suspension was concentration of the peptide that results in the killing of more inoculated into pre-heated Muller-Hinton agar (MHA) than 99.9 % of the tested bacteria (Wise et al. 1983). medium (5 g/l beef infusion solids, 17.5 g/l casein hydrolysate, 1.5 g/l starch, 20 g/l agar, pH 7.4; approximately Bactericidal kinetics assay 42 °C). The medium was then mixed rapidly and poured into a Petri dish. Sterile oxford cups (diameter 6 mm) were Ninety microliters of exponential-phase S. aureus strain placed in the solidified medium, and each cup was filled ATCC 25923 (approximately 104–5 CFU/ml) cells were incu- with 50 μl recombinant P. pastoris supernatant or bated with 10 μl of rMP1106 (final concentrations were 1×, 64 μg/ml purified rMP1106. After incubation at 37 °C 2×, and 4×MIC). The mixed samples were added to the wells for 16–18 h, the zones of growth inhibition were of 96-well cell culture plates (each concentration sample was calculated. performed in quadruplicate) and incubated at 37 °C. One- 2. Antimicrobial titer in the fermentation supernatant was hundred-microliter samples from each well were collected serially diluted twofold with Muller-Hinton broth (MHB) after 0, 2, 4, 6, and 12 h of incubation and were serially diluted medium, and then 10 μl of the diluted samples were added and plated on MHA to count visible colonies. onto solidified MHA medium containing S. aureus strain ATCC 25923 (106 CFU/ml). The titer was defined as the Hemolytic assay reciprocal of the highest dilution (2n) that inhibited bacterial growth. Thus, the activity unit of rMP1106 was The hemolytic activity was evaluated by determining hemo- defined as 2n×1,000 μl/10 μl(Liuetal.2008;Senbagam globin release from erythrocyte suspensions of healthy human et al. 2013). red blood cells. The blood cells were washed three times in

sterile phosphate buffer (100 mM NaCl, 80 mM Na2HPO4, 20 mM NaH2PO4, pH 7.4) and were centrifuged at 1,500 rpm The antimicrobial spectrum assay of rMP1106 for 5 min at room temperature. The 50-μl erythrocyte suspensions (final concentration, approximately 8 %) were then The antimicrobial spectrum of rMP1106 was estimated by an mixed with 50 μl of peptides at different concentrations (2– inhibition zone assay as described by Zhang et al. (2011). All 1,024 μg/ml), and the mixtures were incubated at 37 °C for of the tested strains summarized in Table 2 were grown to an 1 h. PBS and 0.1 % Triton X-100 served as negative and

OD600 nm of 0.4 at 37 °C in MHB medium. Then, 150 μlof positive controls, respectively. Three replicates were per- each cell suspension was inoculated into 15-ml MHA medium formed for each condition. Hemolysis (%)=[(OD540 nm of at approximately 42 °C. Then, sterile oxford cups were placed, the treated sample−OD540 nm of the negative control)/ and rMP1106 (2 μmol), vancomycin (2 μmol), and ampicillin (OD540 nm of positive control−OD540 nm of negative con- (4 μmol) were added to each cup. After incubation at 37 °C trol)]×100 %. overnight, the inhibition zone diameter was measured. Effect of pH, temperature, and serum on the activity MIC and MBC assays of rMP1106

The MIC was determined using a microbroth dilution method, To determine pH value stability, 64 μg of purified peptide was according to the procedures outlined by the Clinical and adjusted to a pH range from 2.0 to 10.0 with the following Laboratory Standards Institute (CLSI). The purified 100 mM buffers: glycine–HCl buffer (pH 2.0), sodium acetate rMP1106 was diluted into twofold serial dilutions with a buffer (pH 4.0), sodium phosphate buffer (pH 6.0), Tris–HCl gradient concentration. The tested strains were grown at buffer (pH 8.0), and glycine–NaOH buffer (pH 10.0). Then,

37 °C in MH medium to an OD600 nm of 0.4 and were diluted the samples were incubated at 37 °C for 4 h. The thermal to a final inoculum concentration of approximately 1× stability of purified rMP1106 was determined after 1-h Appl Microbiol Biotechnol

Table 2 Inhibition spectra of rMP1106 Strain or species Sensitivity Source

rMP1106 Van Amp

Gram-positive Staphylococcus aureus ATCC25923 +++ ++ ++ CVCC S. aureus ATCC6538 + ++ +++ CVCC S. aureus ATCC43300 ++ ++ + CVCC Staphylococcus epidermidis ATCC26069 NZ + ++ CVCC Streptococcus suis CVCC3928 +++ ++ ++ CVCC Streptococcus suis CVCC3309 +++ ++ + CVCC Streptococcus suis CVCC606 +++ ++ ++ CVCC Bacillus licheniformis CMCC1.265 ++ +++ +++ CMCC Bacillus coagulans CMCC1.2407 ++ ++ +++ CMCC Bifidobacterium CMCC1.2212 ++ ++ ++ CMCC Bacillus subtilis ATCC6633 NZ NZ + CVCC Gram-negetive Van vancomycin, Amp ampicillin, Bacterium enteritidis CMCC50336 NZ NZ ++ Yangzhou University NZ no zone, + 10 mm and below, Salmonella pullorum CVCC503 NZ NZ ++ CVCC ++ 11 to 15 mm, +++ 16 mm Salmonella choleraesuis CVCC1802 NZ NZ +++ CVCC and over, CVCC China Veterinary Culture Collection Center, Bei- Salmonella typhimurium ATCC14028 NZ NZ ++ Northeast Agricultural University jing, China, CMCC National Pseudomonas aeruginosa ATCC27853 NZ NZ ++ Northeast Agricultural University Center For Medical Culture col- Escherichia coli K88 NZ NZ NZ CVCC lections, Beijing, China incubation of 64 μg of purified rMP1106 at 4, 20, 40, 60, 80, rMP1106 was dissolved in sterile deionized water, and 10 mM and 100 °C in deionized water, respectively. In addition, the DTT and 20 % DMSO were used as positive and negative rMP1106 (64 μg/ml) was also incubated in fresh human controls, respectively (Peng et al. 2014). serum at 37 °C for 0, 1, 3, 6, and 24 h, and the peptide in sterile deionized water and serum alone were used as controls (Zhu et al. 2012). After treatment, the antimicrobial activity of rMP1106 against S. aureus strain ATCC 25923 was tested using inhibition zone assays. Results

Peptide design Proteolytic digestion resistance assay The positive charge and hydrophobicity are key factors in the The proteolytic digestion assay was performed according to structure and antimicrobial activity of AMPs (Ahn et al. previous reports (Liu et al. 2013;Xietal.2013). Papin (500 U/ 2013). As Table 1 shows, the net charge of MP1106 increased mg, pH 6.0), pepsin (3,000 U/mg, pH 2.0), trypsin (250 U/mg, from +2 to +5.5 and the grand average of hydropathicity pH 8.0), snailase (2,000 U/mg, pH 6.0), and proteinase K (GRAVY) increased from −0.695 to −0.622, indicating that (40 mAU/mg, pH 7.0) solutions were each mixed with MP1106 was more hydrophobic than plectasin. The isoelec- rMP1106 samples at a ratio of 1:10 (w/w)at37°Cfor4h. tric point (PI) of MP1106 was 8.84, and the PI of its parental Untreated peptide solutions were used as positive controls, peptide-plectasin was 7.77. Furthermore, the α-helix of and buffers without peptides were used as negative controls. MP1106 was composed of residues 13–20, and the antiparal- lel β-sheets was composed of residues 27–31 and 36–40, Oxidation and reduction of rMP1106 which was unchanged from plectasin. Keeping the main structure unchanged may help retain the expression levels of Purified rMP1106 (64 μg/ml) was reduced using 5 and 10 mM MP1106. However, the side chains and electron cloud of K11 dithiothreitol (DTT) at 37 °C for 4 h and was oxidized in and H14 extended further than those of plectasin and were aqueous 10 and 20 % dimethyl sulfoxide (DMSO) for 4 h at more separated compared with those of the parental peptide room temperature. The reduced and oxidized rMP1106 were (Fig. 1). Additionally, the side chain of I36 extended further then assessed using the inhibition zone method. The purified than the V36 residue in plectasin, which contributed to the Appl Microbiol Biotechnol

Fig. 1 The simulation of spatial structure of MP1106. a The AB simulated structure of MP1106 based on plectasin. b The structure of plectasin. The α-helix and β-sheets are indicated as pink and yellow, respectively. In addition, four mutated amino acids, their side chains, and the electron cloud are also labeled. White, red,andblue indicates the electron cloud and side chain of aliphatic, carboxyl, and positive charged groups, respectively (color figure online)

stronger hydrophobicity of MP1106. External positive construct the pPICZαA-MP1106 vector (Fig. 2a, b). After line- charged and hydrophobic electron clouds most likely improve arization with PmeI, the pPICZαA-MP1106 vector was trans- its contact with the cell membrane to more effectively elicit ferred into P. pastoris X-33 competent cells by electroporation, the antimicrobial activity of MP1106. and zeocin-resistant P. pastoris transformants were obtained on YPDS containing 100 μg/ml zeocin. Positive transformants Construction of the pPIC ZαA-MP1106 expression vector were screened by PCR (data not shown). and screening of transformants Expression of rMP1106 in shaking flasks and fermentor A codon-optimized MP1106 gene sequence was designed based on the preferential codon usage of yeast (Fig. 2a). The fragment Fifty positive transformants were screened and tested for the was inserted into the XhoIandXbaI sites of pPICZαAto antimicrobial activity of rMP1106. One transformant that produced the maximum diameter of inhibition zone against the S. aureus strain ATCC 25923 was selected. The transformant was further confirmed by inoculation into a shaking flask. Both the inhibition zone diameter and target protein band of collected supernatants increased with induction time (Fig. 3a, b), whereas the target band was not detected in the culture of the transformant harboring the empty plasmid pPICZαA(Fig.3b, lane 1). The total protein concentration in the culture supernatant was 212 mg/l after 120 h of induction in the shaking flask. To further enhance the yield of rMP1106, high-density cultivation was performed in a 5-l fermentor. The target peptide in the supernatant was detected after 24 h of induction, and the concentration increased with induction time (Fig. 4a). The biomass and secreted protein in the culture was up to 317 and 2.134 g/l at 120 h of induction, respectively (Fig. 4d). The rMP1106 was estimated to compose 84.75 % of the total secreted protein and reach 1,808 mg/l after a 120-h induction. Both the inhibition diameter and activity unit increased with induction time (Fig. 4b, c), and the activity unit reached 51,200 AU/ml after a 120-h induction. Fig. 2 The construction of the pPICZαA-MP1106 plasmid. a The MP1106 nucleotide sequence and its corresponding amino acid sequence. Purification of rMP1106 The underlined letters are the XhoIandXbaI restriction endonuclease sites, and the arrows indicate the cleavage sites. The underlined lower- case letters indicate the Kex2 cleavage site. b A schematic diagram of the The rMP1106 was purified by a SP Sepharose FF cation- recombinant expression vector pPICZαA-MP1106 exchange column in a one-step elution. The highest peak Appl Microbiol Biotechnol

Fig. 3 Expression of rMP1106 in P.pastoris X-33 in shaker flask level. a analysis of rMP1106 in fermentation supernatants. Lane M,atotalof5μl Antimicrobial activity of fermentation supernatants in different induction of protein molecular weight marker. Lane 1,10μl of 120-h fermentation times against the S. aureus strain ATCC25923. Numbers 1, 2, 3, 4, 5,and supernatants from P. pastoris X-33 contained pPICZαA plasmid. Lanes 6, a total of 50-μl fermentation supernatants taken at 0, 24, 48, 72, 96, and 2–7,10μl of rMP1106 fermentation supernatants taken at 0, 24, 48, 72, 120 h, respectively. Number 7,3μg vancomycin. b Tricine-SDS-PAGE 96, and 120 h of induction, respectively absorbance fractions eluted by 600 mM NaCl were pooled the purified sample was obtained (Fig. 5d), which is consistent and detected as a single band by Tricine-SDS-PAGE without with its theoretical value of 4,410.12 Da (Table 1). any other unspecific bands (Fig. 5a, c). The purified rMP1106 showed antimicrobial activity to the S. aureus strain ATCC Antimicrobial spectrum of rMP1106 25923, and other samples showed only weak antimicrobial activity (Fig. 5b). The recovery of rMP1106 was estimated to rMP1106 showed a potent antimicrobial spectrum against be 46.96 %, and its yield reached 831 mg/l with 94.68 % some Gram-positive bacteria, including S. aureus, Streptococ- purity. The MALDI-TOF MS analysis indicated that only one cus suis, Bacillus licheniformis, Bacillus coagulans,and target peak with a major molecular mass of 4,409.8 Da from Bifidobacterium. However, rMP1106 could not inhibit

A B M 1 2 3 4 5 6

1 234

5 6 78

5.8 kDa

3.3 kDa 9 10 11 12

Fig. 4 Expression of rMP1106 in P. pastoris X-33 in 5-l fermentor. a rMP1106 fermentation supernatant after induction for 0, 24, 48, 72, 96, Tricine-SDS-PAGE analysis of rMP1106 in fermentation supernatants of and 120 h. Circle 12,3μg vancomycin. c The activity units of recombi- P. pastoris using high-density cultivation. Lane M,5μl of protein nant P. pastoris-rMP1106 fermentation supernatant after induction for 0, molecular weight marker; lanes 1–6, supernatants (10 μl each) harvested 24, 48, 72, 96, and 120 h. d Time curve of the concentration of secreted at 0, 24, 48, 72, 96, and 120 h of induction, respectively. b Antimicrobial protein levels and cell wet weight during induction. Triplicate observa- activity of recombinant P. pastoris fermentation supernatant against tions were made, and data are presented as the means±standard error S. aureus ATCC25923. Circles 1–11,30μl recombinant P. pastoris- Appl Microbiol Biotechnol

Fig. 5 Purification and A identification of rMP1106. a Purification of rMP1106 by a SP sepharose FF cation-exchange 1 2 3 4 column. a The purple and blue B lines represent the peak monitored by measuring UV absorbance at 215 and 280 nm, respectively. b Antimicrobial activity of unpurified sample and pooled protein at the highest peak absorbance fractions against the M 1 2 3 4 S. aureus strain ATCC25923. C Circle 1,50μl unpurified sample; Circles 2–4,50μl pooled protein at peaks 1–3. c Tricine-SDS- 5.8 kDa PAGE analysis of unpurified sample and pooled protein at 3.3 kDa the highest peak absorbance fractions. Lane M,5μlofprotein molecular weight marker; lanes Peak 1 Peak 2 Peak 3 2–4,10μl of pooled protein at peaks 1–3. d MALDI-TOF MS D analysis of the purified rMP1106 (color figure online)

Gram-negative bacteria in any tested strains (Table 2). It did ATCC25923, ATCC29213, and ATCC43300, but not not change the antimicrobial spectrum compared with for S. aureus strain ATCC6538 (0.45 vs 0.08). Com- plectasin (Mygind et al. 2005; Schneider et al. 2010). The pared with the parental peptide plectasin (0.11 μM; characteristic of rMP1106 against Streptococcus suis is stud- 7.27 μM), rMP1106 (0.014 μM; 0.91 μM) had higher ied in another paper in press. This work focuses on the antimicrobial activity against ATCC 25923 and ATCC property of rMP1106 against S. aureus, including MSSA 43300 (MRSA) and lower activity against ATCC 29213 and clinical MRSA strains. and ATCC6538 (Table 3). Due to the high activity of rMP1106 to the tested MRSA Antimicrobial activity of rMP1106 ATCC standard strain, 20 clinical MRSA strains were also tested to evaluate the antimicrobial activity of rMP1106 (Ta- The rMP1106 exhibited a strong antimicrobial activity against ble 4). The results showed that rMP1106 was active against the tested strains at low MICs from 0.014 to 0.91 μM(Ta- these clinical MRSA strains with low values of MICs from ble 3). Compared with traditional antibiotics, the activity of 0.014 to 1.8 μM and MBCs from 0.06 to 3.64 μM, which was rMP1106 (0.014–0.91 μM) was stronger than ampicillin stronger than the traditional antibiotics ampicillin (MICs from (1.35–10.8 μM) to three of the tested strains of S. aureus 11.46 to 366.76 μM) and vancomycin (MICs from 0.67 to

Table 3 The MICs and MBCs of rMP1106 against S. aureus Strains MIC (μM) MBC (μM)

MP1106 Van Amp Plectasina MP1106 Van Amp

ATCC25923 0.014 0.08 1.35 0.11 0.056 0.08 1.35 ATCC29213 1.8 0.33 2.7 0.23 7.2 1.32 10.8 ATCC6538 0.45 0.33 0.08 0.23 0.45 0.33 0.08 a Data cited from previous results ATCC43300 0.91 0.33 10.8 7.27 3.6 1.32 21.6 (Mao et al. 2013) Appl Microbiol Biotechnol

Table 4 MIC and MBC assays of rMP1106 against MRSA clinical MRSA MIC (μM) MBC (μM) isolates MP1106 Plectasina Ampa Vana MP1106 Ampa Vana

20009 0.03 0.73 11.46 0.67 0.12 45.84 1.33 20010 0.06 1.46 22.92 0.67 0.24 91.69 0.67 20013 0.06 1.46 183.38 0.67 0.12 366.76 0.67 20015 0.03 2.91 91.69 0.67 0.06 366.76 0.67 20018 0.03 0.73 366.76 0.67 0.06 >366.76 1.33 20023 0.03 0.73 366.76 1.33 0.12 >366.77 2.67 20024 0.03 0.73 45.84 0.33 0.12 91.69 0.33 20027 1.8 1.46 183.38 0.67 1.8 >366.76 2.67 20032 0.06 0.73 45.84 0.67 0.24 183.38 2.67 20033 0.06 0.73 183.38 1.33 0.12 366.76 1.33 20035 0.06 0.73 91.69 1.33 0.12 366.76 2.67 20040 0.03 1.46 91.69 0.67 0.12 366.76 0.67 20045 0.06 1.46 91.69 0.67 0.24 366.76 0.67 20047 0.03 0.73 183.38 0.67 0.13 >366.76 0.67 20049 0.91 1.46 366.76 2.67 3.64 >366.76 5.33 a Data cited from previous results 20055 0.014 1.46 183.38 0.33 0.028 366.76 1.33 (Xi et al. 2013; Zhang et al. 2014). 20056 0.91 1.46 183.38 1.33 3.64 366.76 2.67 Clinical strains were offered by the Department of Food Science 20057 1.8 0.73 366.76 1.33 3.6 >366.76 1.33 and the Bor Luh Food Safety 20064 0.014 1.46 183.38 1.33 0.056 366.76 2.67 Center, Shanghai Jiao Tong Uni- SJU05P157 0.23 1.46 11.64 1.33 0.92 45.84 1.33 versity (SJU)

2.67 μM). Compared with the parental peptide plectasin, Effect of pH, temperature, serum, and proteinases rMP1106 was more active against the most tested strains on rMP1106 except for 20027 and 20057 (Table 4). Purified rMP1106 retained a stable activity level against the S. aureus strain ATCC25923 in the temperature range of 20 to Bactericidal kinetics assay 40 °C, and 80 % of the activity remained after a 1-h incubation at 100 °C (Fig. 8a). Meanwhile, rMP1106 exhibited stable The time-killing kinetics curve showed that the counts of activity against the S. aureus strain ATCC 25923 in neutral

S. aureus strain ATCC 25923 (log10 CFU/ml) steadily and alkaline environments (pH 6, 8, and 10), and it was increased to 6.32 with medium alone. The treatment with slightly reduced in an acidic environment (pH 2 and 4) 2× and 4× MIC of rMP1106 resulted in a sustained (Fig. 8b). rMP1106 also showed a strong serum stability, even reduction in the number of bacterial counts from 6.32 to 2.94 and 3.00, respectively. The antimicrobial effect was 7 better than 2× MIC vancomycin (log10 CFU/ml was 3.8 in CK MIC 2MIC 4MIC 2Van 6 h). However, regrowth of S. aureus strain ATCC25923 6 wasobservedafter6hoftreatingwith1×MICrMP1106 5

(Fig. 6). (CFU/mL) 10 4 Log

3 Hemolytic assay 2 024612 Different concentrations of rMP1106 (0 to 512 μg/ml) were Time (h) tested to observe its lysis activity on human red blood cells Fig. 6 Killing kinetics of rMP1106 against the S. aureus strain (RBCs) in hemolysis assays (Fig. 7). At the highest concen- ATCC25923 in vitro. CK, S. aureus ATCC 25923 was incubated in the tration of 512 μg/ml, the hemolysis was only 1.16 %, indicat- presence of medium alone; MIC, 2× MIC, 4× MIC, the S. aureus strain ATCC 25923 was incubated in the presence of purified rMP1106 at 1×, ing that rMP1106 had little or no hemolytic activity to the 2×, or 4× MIC, respectively; 2Van, the S. aureus strain ATCC 25923 was human RBCs. incubated in the presence of vancomycin at 2× MIC Appl Microbiol Biotechnol

10 when incubated in serum for 24 h (Fig. 8d). rMP1106 was 9 resistant to pepsin, papain, and protein K and highly sensitive 8 to trypsin (approximately 46 % of its activity was lost) 7 (Fig. 8c). 6 5 4

Hemolysis(%) 3 Effect of oxidation and reduction on the activity of rMP1106 2 1 As shown in Fig. 9, the inhibition zone diameters of oxidized 0 rMP1106 with 10 and 20 % DMSO were nearly the same size 01248163264128256512 as control. However, the inhibition zone diameters of reduced rMP1106 (µg/ml) rMP1106 were smaller than the control (13 vs 15 mm). These Fig. 7 Hemolytic activity of rMP1106 against human erythrocytes. results suggested that DTT treatment has a minor impact on Hemolytic activity was determined by measuring the hemoglobin absor- the antimicrobial activity of rMP1106. bance at OD540 nm in supernatants. Three duplicate observations were made; bars represent the standard error of the mean

Fig. 8 The effects of temperature, pH, and serum on the activity of was treated with papain, pepsin, trypsin, snailase, and proteinase K and rMP1106 and its resistance to proteinases. a Effects of temperature on incubated at 37 °C for 4 h, and rMP1106 in the proteinases’ optimal the antibacterial activity of purified rMP1106. Purified rMP1106 was buffers and buffers alone were used as control. d Effects of human serum incubated at 4, 20, 40, 60, 80, and 100 °C for 1 h. b Effects of pH on on the antibacterial activity of purified rMP1106. Purified rMP1106 as the antibacterial activity of purified rMP1106. Purified rMP1106 was incubated in human serum or sterile deionized water at 37 °C for 0, 1, 3, 6, incubated in buffers with pH values of 2.0, 4.0, 6.0, 8.0, and 10.0 at and 24 h. All of the experiments were performed in triplicate; bars 37 °C for 4 h. c Resistance to proteinases of rMP1106. Purified rMP1106 represent the standard error of the mean Appl Microbiol Biotechnol

Discussion 2013). Sequentially, the V36 was mutated to an I residue to enhance the hydrophobicity of MP1106. Moreover, the exter- AMPs were first characterized by their antimicrobial proper- nal side chains and electron cloud of K11 and H14 (Fig. 1) ties, and some AMPs have also been shown to have immuno- most likely enhance the binding of MP1106 to the negatively regulatory effects (Auvynet and Rosenstein 2009). Currently, charged phospholipid bilayer of bacteria. The exact structure 2411 AMPs are registered in the antimicrobial peptide data- and mechanism of MP1106 remain to be elucidated. base (APD) (http://aps.unmc.edu/AP/main.php). However, This work has shown that rMP1106 has a narrow spectrum only a few of them advanced into clinical trials (Hancock of antimicrobial activity against some Gram-positive bacteria, and Sahl 2006; Yeung et al. 2011;Zasloff2002). The use of similar to the activities of plectasin and NZ2114 (Hara et al. AMPs was primarily hampered by their poor antimicrobial 2008; Ostergaard et al. 2009). However, rMP1106 can also activity, stability, and toxicity. In recent years, a large number inhibit probiotics such as B. licheniformis, B. coagulans,and of studies have aimed to overcome these setbacks by using Bifidobacterium, which resulted from the fact that plectasin self-assembling peptide nanoparticles and peptides containing exhibits its antibacterial effects by directly binding Lipid II, unnatural amino acids and hybrid peptides (Giuliani et al. the essential precursor for the cell wall of Gram-positive 2008; Liu et al. 2009;Tianetal.2009b). In addition, the bacteria such as Bacillus and Staphylococcus (Schneider optimization of some native AMP sequences based on posi- et al. 2010). In addition, there was an eightfold increase in tive net charge and hydrophobicity have also been tested. antimicrobial activity against the S. aureus strain ATCC43300 NZ2114 was designed by optimizing the amino acid sequence (MRSA) compared with plectasin (0.9 vs 7.27 μM, respec- of plectasin with three mutational sites (D9N, M13L, and tively) (Table 3), and rMP1106 showed a stronger antimicro- Q14R), and these mutations improved bactericidal activity bial activity against MRSA (Table 3) than ampicillin and against Staphylococci (Ostergaard et al. 2009; Raventos vancomycin. As a result, rMP1106 was highly active against et al. 2005). 20 clinical MRSA strains and exhibited a stronger antimicro- Positive net charge and hydrophobicity are the two main bial activity than typical antibiotics and plectasin (Table 4). factors required for the antimicrobial activity of AMPs Furthermore, it killed approximately 90 % the S. aureus strain (Powers and Hancock 2003). AMPs with positive net charges ATCC 25923 within 4 h in 2× and 4× MIC, which was more bind to the negatively charged surface of bacteria through effective than vancomycin treatment in 2× MIC (Fig. 6). electrostatic bonding, which is thought to be the first step in These results suggested that rMP1106 has potential as a potent promoting the interaction between AMPs and cell mem- agent against S. aureus,especiallyMRSAstrains. branes. Several magainin analogs with different charges (0 The high manufacturing costs has been another roadblock to +6) were synthesized, and an increase in positive charge in the clinical implementation of AMPs (Findlay et al. 2010). enhanced the bactericidal activity of the analogs (Matsuzaki Majority of directly expressed AMPs, such as human α- et al. 1997). MSI-78 was designed as a synthetic analog to defensin (HD5) and enterocin L50 (Parachin et al. 2012), were magainin 2 by increasing its positive charge and stabilizing expressed in P. pastoris. In our previous works, plectasin and the amphipathic structure. MSI-78 exhibited excellent antimi- its derived peptide NZ2114 were successfully expressed in crobial activity and has been developed in clinical trials for the P. pastoris with high yields (Zhang et al. 2011, 2014). In this treatment of diabetic foot ulcers (Maloy and Kari 1995). Although it was abandoned in phase III, the strategy of increasing positive net charge was also applied to the modifications of AMPs to develop therapeutic agents with more potent antimcrobial activity against pathogens (Hancock 1997). In this study, two acidic amino acids, D11 and Q14, of plectasin were replaced by cationic amino acids K and H, respectively, which increased the positive charge of the parental peptide to +5.5, improving the antimicrobial activity of the parental peptide. In addition, these modifications may also alter the antimicrobial mechanism of MP1106 to two ways by affecting Lipid II binding and membrane disruption. Hydrophobicity is another parameter for the antimicrobial activity of AMPs. Previous studies have shown that LB-PG and CA-PG, which are hybridized by progetrin-1, lactoferricin, and cecropin A and which are more hydrophobic compared to their parental Fig. 9 Oxidation and reduction of rMP1106. 1, 20 % DMSO alone; 2, peptides, significantly broadened their antimicrobial activity 10 mM DTTalone; 3 and 4, rMP1106 treated with 10 and 20 % DMSO; 5 against pathogens and reduce hemolytic activity (Liu et al. and 6, rMP1106 treated with 5 and 10 mM DTT; 7,rMP1106alone Appl Microbiol Biotechnol study, MP1106 was also successfully expressed in P. pastoris environments, it showed very low hemolysis in human RBCs, X-33 with 1,808 mg/l in a 5-l fermentor, which was higher high stability over a wide range of temperatures and was quite than that of plectasin (748.63 mg/l of total secreted protein) stable in human serum. All of the above results indicate that and NZ2114 (860 and 1,309 mg/l in the total secreted protein rMP1106 is a potential candidate for further development as a at 29 and 25 °C, respectively). This yield is also higher than therapeutic drug. most AMPs produced by P. pastoris previously (Parachin et al. 2012). In addition, rMP1106 was purified by a one- Acknowledgments The authors wish to acknowledge Prof. Zhu step method with an ion-exchange column. The high level of Guoqiang (College of Veterinary Medicine, Yangzhou University) for kindly providing Bacterium enteritidis CMCC50336 and Prof. Shan expression and straightforward purification method highlight Anshan (Insitute of Animal Nutrition, Northeast Agricultural University) rMP1106 as a convenient product for industrial production. for kindly providing Salmonella typhimurium ATCC14028 and Pseudo- The toxicity and stability of AMPs limit their application in monas aeruginosa ATCC27853. We gratefully acknowledge Prof. Yang clinical treatments (Eckert 2011). Studies have shown that Fuquan, Ph.D., in the Proteomics Platform Laboratory at the Institute of Biophysics, Chinese Academy of Sciences, for his coordination of the plectasin and NZ2114 exhibit no hemolysis in rabbit and MALDI-TOF MS analysis. This study was supported by the National human RBCs (Yang et al. 2011; Zhang et al. 2014). In this Natural Science Foundation of China (No. 31372346 and No. 31302004), work, rMP1106 also exhibited a low hemolytic level and the Project of the National Support Program for Science and Technology caused the lysis of only 1.16 % human RBCs even at a high in China (No. 2013BAD10B02 and No. 2011BAD26B02), the Special μ Fund for Agro-scientific Research in the Public Interest in China (No. concentration of 512 g/ml (Fig. 7). Stability assays showed 201403047), and the AMP Direction of National Innovation Program of that rMP1106 could endure high temperatures even at 100 °C Agricultural Science and Technology in CAAS (2013–2017). for1h(Fig.8a), and rMP1106 also showed a higher activity in neutral and alkaline environments but a slightly reduced activity in acidic environments (Fig. 8b). However, plectasin References remained stable over a range from pH 2.0 to 10.0 (Zhang et al. 2011). The reduced tolerance to acidic environments might be Ahn M, Murugan RN, Jacob B, Hyun JK, Cheong C, Hwang E, Park HN, due to the replacement of its two acidic amino acids Asp11 Seo JH, Srinivasrao G, Lee KS, Shin SY,Bang JK (2013) Discovery and Gln14 by Lys and His. 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