Comparative Analysis of the Antimicrobial Activities of PlantDefensin-Like and Ultrashort Peptides against Food-Spoiling Bacteria

Joanna Kraszewska, Michael C. Beckett, Tharappel C. James, Ursula Bond

Microbiology Department, Moyne Institute, School of Genetics and Microbiology, Trinity College Dublin, Dublin, Ireland

ABSTRACT

Antimicrobial peptides offer potential as novel therapeutics to combat food spoilage and poisoning caused by pathogenic andnonpathogenic bacteria. Our previous studies identified the peptide human beta-defensin 3 (HBD3) as a potent antimicrobialagent against a wide range of beer-spoiling bacteria. Thus, HBD3 is an excellent candidate for development as an additive to pre-vent food and beverage spoilage. To expand the repertoire of peptides with antimicrobial activity against bacteria associated withfood spoilage and/or food poisoning, we carried out an in silico discovery pipeline to identify peptides with structure and activ-ity similar to those of HBD3, focusing on peptides of plant origin. Using a standardized assay, we compared the antimicrobialactivities of nine defensin-like plant peptides to the activity of HBD3. Only two of the peptides, fabatin-2 and Cp-thionin-2, dis-played antimicrobial activity; however, the peptides differed from HBD3 in being sensitive to salt and were thermostable. Wealso compared the activities of several ultrashort peptides to that of HBD3. One of the peptides, the synthetic tetrapeptide O3TR,displayed biphasic antimicrobial activity but had a narrower host range than HBD3. Finally, to determine if the peptides mightact in concert to improve antimicrobial activity, we compared the activities of the peptides in pairwise combinations. The plantdefensin-like peptides fabatin-2 and Cp-thionin-2 displayed a synergistic effect with HBD3, while O3TR was antagonistic. Thus,some plant defensin-like peptides are effective antimicrobials and may act in concert with HBD3 to control bacteria associatedwith food spoilage and food poisoning.

IMPORTANCE

Food spoilage and food poisoning caused by bacteria can have major health and economic implications for human society. Withthe rise in resistance to conventional antibiotics, there is a need to identify new antimicrobials to combat these outbreaks in ourfood supply. Here we screened plant peptide databases to identify peptides that share structural similarity with the human de-fensin peptide HBD3, which has known antimicrobial activity against food-spoiling bacteria. We show that two of the plant pep-tides display antimicrobial activity against bacteria associated with food spoilage. When combined with HBD3, the peptides arehighly effective. We also analyzed the activity of an easily made ultrashort synthetic peptide, O3TR. We show that this small pep-tide also displays antimicrobial activity against food-spoiling bacteria but is not as effective as HBD3 or the plant peptides. Theplant peptides identified are good candidates for development as natural additives to prevent food spoilage.

Antimicrobial peptides (AMPs) are short oligopeptides rang-ing from 4 to 100 amino acids that display antimicrobial ac-

tivity against a broad range of pathogens, including Gram-positiveand -negative bacteria, fungi, viruses, protists, parasites, and eveninsects (1). AMPs form part of the innate immune response, act-ing as a first line of defense and assisting in the rapid elimination ofinvading pathogens (2). The peptides can also invoke adaptiveimmune responses as a secondary response (3). The host rangeand specificity of antimicrobial activity are unique to each peptide.

AMPs can be linear or cyclic and are generally grouped intospecific classes based on their biological and physical properties.The major classes of peptides are (i) anionic, (ii) cationic, (iii)cationic and amphipathic, and (iv) �-helical (4, 5). More than5,000 AMPs have been identified from a wide range of organisms,including bacteria, plants, invertebrates, and vertebrates, throughbiochemical characterization of purified peptides or through insilico analyses of peptides based on gene sequence or on structuraland/or physicochemical similarities to known AMPs (6–11). Ra-tional combinatorial peptide design, coupled with high-through-put screening, has also been employed to design novel syntheticAMPs (12).

The majority of AMPs elicit antimicrobial activity through in-teraction with cellular membranes, and several models have been

proposed to describe their mode of action (13). The barrel-stavemodel proposes that positively charged peptides interact with thecellular membranes through interaction with polar phospholipidhead groups. Once a critical concentration is reached, the peptidesself-aggregate and are inserted into the membrane perpendicu-larly to create transmembrane pores lined with peptides, resultingin the disruption of ionic and proton gradients (14). A relatedmodel, the toroidal model, hypothesizes that at a critical concen-tration of peptides, the strain induces membranes to curve in-ward, creating donut-like pores lined with peptides and phospho-

Received 22 February 2016 Accepted 2 May 2016

Accepted manuscript posted online 6 May 2016

Citation Kraszewska J, Beckett MC, James TC, Bond U. 2016. Comparative analysisof the antimicrobial activities of plant defensin-like and ultrashort peptidesagainst food-spoiling bacteria. Appl Environ Microbiol 82:4288 –4298.doi:10.1128/AEM.00558-16.

Editor: J. Björkroth, University of Helsinki

Address correspondence to Ursula Bond, ubond@tcd.ie.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.00558-16.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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lipid head groups (15). An alternative model, the carpet model,proposes that small areas of membrane become coated with pep-tides, creating hydrophilic pockets, which disrupt the lipid bilayer(16).

Besides interacting with the membrane, some peptides exertantimicrobial activity through interacting with other cellular tar-gets, inhibiting cellular processes such as nucleic acid and proteinsynthesis and disrupting multienzyme complexes such as the elec-tron transport chain and cell wall synthesis complexes (17–19).

One of the most important categories of AMPs is the defensins,a class of small cationic, amphipathic peptides that have beenidentified in both vertebrates and invertebrates (20). Like othercationic peptides, defensins rapidly and efficiently permeabilizemembrane bilayers (21, 22) and also appear to inhibit bacterial cellwall biosynthesis (19, 23). In mammals, the peptides are producedin cells and tissues such as salivary glands and Paneth cells and aresecreted into tissues that come into contact with invading mi-crobes, for example, the buccal cavity and the intestinal lining (24,25). Defensins are also produced in abundance in plants, mainlyin seeds, where they are constitutively expressed in storage organsor are induced either systemically or locally in response to attackby pathogenic microorganisms (26, 27).

Defensins generally range from 45 to 54 amino acids, and al-though the peptides differ greatly at the amino acid sequence level,they possess many conserved structural and biochemical features,such as twisted antiparallel beta-sheets, a short amphipathic �-helix, conserved cysteine residues, an overall positive netcharge, and a net negative hydrophobicity score (28). Based onthe pattern of intramolecular disulfide bonds formed by the cys-teine residues, defensins are classified as alpha- or beta-defensins(29). A third type of cyclic defensins, named theta, has so far beenfound only in Old World rhesus macaque monkeys (30). Whilebeta-defensins are found in vertebrates, invertebrates, and plants,alpha-defensins are confined to mammals and generally lack theshort �-helix (31). Defensins can have broad or narrow antimi-crobial activity and are effective against Gram-positive and -neg-ative bacteria, fungi, and some viruses.

The prototypical defensin is human beta-defensin-3 (HBD3;encoded by the DEFB103A gene), a broad-spectrum antimicro-bial with activity against Gram-positive and -negative bacteria.The peptide is either expressed constitutively or induced uponbacterial challenge (32). Due to its broad specificity, HBD3 is agood candidate for development as an antimicrobial agent, par-ticularly in light of the increasing instances of bacterial resistanceto conventional antibiotics.

We recently demonstrated that HBD3 displays antimicrobialactivity against beverage-spoiling bacteria, such as Lactobacillus,Pectinatus, and Pediococcus spp., and that when expressed in situ inyeasts, HBD3 can provide protection against beer-spoiling bacte-ria during industrial fermentation (33). To expand the repertoireof peptides with antimicrobial activity against bacteria associatedwith food spoilage and food poisoning, we carried out an in silicoscreen to search for peptides with structures and activities similarto those of HBD3. Since plants produce and store in seeds a wideand diverse group of antimicrobial peptides, we initially focusedour analysis on defensin-like peptides of plant origin. We alsoincluded in our study several nondefensin AMPs native to plantsand/or of a synthetic origin. The antimicrobial activities of thepeptides against a range of Gram-positive and -negative food-

spoiling bacteria were examined in parallel and were compared tothat of the prototype, HBD3.

MATERIALS AND METHODSPeptide discovery and bioinformatic analysis. Potential candidate anti-microbial peptides from plants were mined from the PhytAMP database(http://phytamp.pfba-lab-tun.org/main.php), which contains informa-tion on 273 plant antimicrobials. The database was queried for defensinand defensin-like molecules that had been shown previously to have an-timicrobial activity. Peptide sequences for the candidate peptides identi-fied were retrieved from the UniProt database (http://www.uniprot.org/),and any containing the “knottin” peptide motif and scorpion toxin-likemotifs were rejected. Where structures were available, they were retrievedfrom the Protein Data Bank server (http://www.rcsb.org/pdb/home/home.do). Candidate peptides with no experimentally determined struc-ture were modeled using the protein structure prediction program Ro-betta (http://robetta.bakerlab.org/). All modeling was conducted usingdefault protocols and parameters (34, 35). Template structures againstwhich modeling was conducted were identified using the Ginzu PDB tem-plate identification and domain prediction protocol. Templates for mod-eling were chosen using an all-atom energy-based selection process.Structures were modeled using the Structure Prediction Tool (robetta.bakerlab.org; Domain Parsing & 3-D Modeling service), set to defaultparameters. The template and alignment most enriched in the lowest-energy population of all-atom refined models was then selected.

Candidate peptide structures were aligned to, and compared with, theempirically determined structure of human beta-defensin 3 (HBD3; PDBidentifier [ID] 1KJ6). Alignment was carried out separately against thehelix and beta-sheet regions of the peptides. Alignment root mean squaredeviation (RMSD) scores of 9.0 for the beta-sheet region and 4.5 for thehelix region were used as cutoffs. The peptides were visualized using thePyMOL software suite (PyMOL Molecular Graphics System, version1.7.4; Schrödinger, LLC). The distribution of charged and hydrophobicresidues of the candidate peptides was visually compared to that ofHBD3. Peptide net charge, hydrophilicity, and isoelectric point werecalculated using Bachem’s and Innovagen’s peptide property calcula-tion tools. The average hydrophobicity was calculated using the onlineGRAVY (grand average of hydropathy) tool (http://www.gravy-calculator.de/index.php) or was determined manually (36).

Peptide synthesis. Synthetic antimicrobial peptides were chemicallysynthesized at a purity of �70% (GL Biochem Ltd., Shanghai, People’sRepublic of China). All peptides contain a C-terminal amide group. Forsome peptides, an acetyl group (CH3CO) was added to the N terminus.The peptides were dissolved in 100 mM acetic acid at 20 mg ml�1 and werestored frozen at �70°C. The stocks were diluted in phosphate-bufferedsaline (PBS, comprising 10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mMNaCl, and 2.6 mM KCl [pH 7.4]; Oxoid, Ltd., Basingstoke, United King-dom) or the appropriate buffer immediately before use.

Strains and growth conditions. Lactobacillus brevis L1055 and Pedio-coccus (Lactobacillus) dextrinicus DSM20293 (from the Deutsche Sam-mlung von Mikroorganismen and Zellkulturen, Leibniz Institute, Braun-schweig, Germany) were grown in MRS broth (Lab M Ltd., Lancashire,United Kingdom) at 30°C under aerobic and microaerobic conditions,respectively. Staphylococcus aureus strain Newman, Salmonella entericasubsp. enterica serovar Typhimurium SL1344, Escherichia coli MG1655,and Pseudomonas aeruginosa NCIMB 950, sourced from the Moyne Insti-tute Culture Collection, Trinity College Dublin, Dublin, Ireland, weregrown under aerobic conditions at 37°C in tryptic soy broth (TSB; SigmaChemical Co., Poole, United Kingdom). Bacillus cereus NCIMB 1526(Moyne Institute Culture Collection) was grown under aerobic condi-tions at 30°C in L broth (Thermo Fisher Scientific, MA, USA). Saccharo-myces cerevisiae strain S-1502B was cultured under aerobic conditions inyeast extract-peptone-dextrose (YEPD) broth at 30°C (37).

Antimicrobial activity assay. The antimicrobial activities of selectedpeptides were assessed using a standard assay for defensin (33, 38, 39).

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Unless otherwise stated, all media and solutions were prewarmed to thepermissive temperature for strain growth. Bacterial cultures were dilutedto an optical density at 600 nm (OD600) of �0.1 to 0.2 in 10 ml of mediumand were grown to mid-exponential phase (OD600, �0.5). The cultureswere diluted in PBS to a final concentration of 2.0 � 103 to 4.5 � 103 cellsml�1. An initial high-throughput spectrophotometry-based screening as-say was conducted as follows. AMPs, at various concentrations, wereadded to 96-well plates in 50 �l of PBS. To this 50 �l, bacteria were added.As a control, the bacterial cultures were added to PBS with no addedpeptides. The microtiter plate was incubated for 1.5 h at either 30°C or37°C depending on the strain growth conditions. Postchallenge, 100 �l of2� medium was added to each well. The plates were incubated at thestrain permissive temperature, and the absorbance at a � of 540 nm wasmeasured immediately (0 h) and following a 24-h incubation for Staphy-lococcus aureus Newman, Salmonella enterica, E. coli, Pseudomonas aerugi-nosa, and Bacillus cereus 1526, after 48 h for Lactobacillus brevis, and after72 h for Pediococcus (Lactobacillus) dextrinicus DSM20293.

For a more quantitative assessment of AMP activity, a spread plateassay was performed as follows. Briefly, 100 �l of AMPs, at various con-centrations in prewarmed PBS, were mixed in a 96-well microtiter platewith 100 �l of L. brevis, diluted to a concentration of 4.5 � 103 cells ml�1

in prewarmed PBS. As a control, the diluted bacterial culture was added toprewarmed PBS with no added peptides. Following incubation for 1.5 h at30°C, 100 �l from each well was spread onto an MRS agar plate. Survivingcolonies were counted after 48 h of incubation at 30°C. To exclude any lossof viability or bacterial growth during the in vitro assay, bacterial CFUwere measured immediately after dilution in PBS (without added pep-tides) and after incubation for 1.5 h. No observed growth or loss of via-bility was observed during the 1.5 h of incubation in PBS in the absence ofadded peptides (data not shown). Bacterial survival following AMP treat-ment was assessed relative to the survival of untreated control samples andwas expressed as a percentage of control survival, taken as 100%. Theconcentration of a peptide required to inhibit 50% or 95% of bacteria(IC50 or IC95, respectively) under the experimental conditions was deter-mined.

To examine the thermostability of AMPs, the peptides, dissolved inPBS to the IC95 in a volume of 150 �l, were first incubated at 100°C for 10min and then allowed to cool for 30 min at a controlled ambient temper-ature (25°C) prior to use in the AMP assay. Alternatively, the peptidesolution was rapidly cooled by plunging it into an ice bath at 4°C. Like-wise, the influence of the salt concentration on antimicrobial activity wasassessed by conducting the AMP assay in phosphate buffer (PB, compris-ing10 mM Na2HPO4, 1.8 mM KH2PO4, and 2.6 mM KCl [pH 7.4]) con-taining different NaCl concentrations (0 to 150 mM). Bacterial survivalwas enumerated following incubation on MRS agar plates for 48 h.

Kinetics of action and synergism of peptides. The kinetics of actionof the antimicrobial peptides were determined as follows. Briefly, 100 �lof AMPs at the IC95 was incubated with 100 �l of L. brevis at a concentra-tion of 4.5 � 103 cells ml�1 for increasing time intervals. Bacterial survivalwas enumerated following incubation on MRS agar plates for 48 h.

To examine the effects of combinations of peptides on bacterial sur-vival, the assay was repeated using AMPs in different combinations at�IC50 or IC95. Bacterial survival was enumerated following incubation onMRS agar plates for 48 h. A combination index (CI) score was calculatedfor each combination of peptides, using the time required to achieve IC50

to IC90 as the measured effect. The CI score was calculated using theformula [(time to reach ICn with peptide 1� peptide 2)/(time to reach ICn

with peptide 1)] � [(time to reach ICn with peptide 1� peptide 2)/(timeto reach ICn with peptide 2)]. A CI score of 1.0 indicates synergisticactivity of the pair of peptides, and a CI score of 1.0 indicates additiveeffects, while a CI score of 1.0 indicates antagonistic effects (40).

Statistical analysis. Experiments were conducted using three techni-cal replicates and were repeated on at least two to three independentoccasions unless otherwise stated. The data were analyzed using MicrosoftExcel and GraphPad Prism (version 5.0) software. P values were deter-

mined using a Student t test or one-way ANOVA (analysis of variance)statistical tests. P values of 0.05 were considered to be statistically sig-nificant.

RESULTS

More than 350 defensins and defensin-like peptides have beenidentified to date and have been designated defensins based onstructural characteristics, antimicrobial activities, and biochemi-cal characterization (41). Most of these peptides have been char-acterized individually, but rarely have they been directly com-pared to each other or their combined effects assessed in a definedassay. The reported antimicrobial specificities and levels of effec-tiveness of the peptides often differ from study to study, largelydue to the variety of methods used to determine inhibitory con-centrations (IC) or lethal concentrations (LC) of the peptides.Furthermore, the lack of conservation at the primary sequencelevel hinders the discovery of new defensin peptides.

To expand the repertoire of peptides with antimicrobial activ-ity against bacteria associated with food spoilage and/or food poi-soning, we undertook an initial selection screen to identify pep-tides with structures and activities similar to those of HBD3. Thescreening criteria were as follows: (i) the peptides were of plantorigin; (ii) peptides containing undesirable motifs associated withcytotoxic and toxin activities were excluded; (iii) the predictedstructures of the peptides were compared to that of HBD3; and(iv) antimicrobial activity and host range, as determined in previ-ous studies, were scored. Initially, 52 plant defensin-like peptideswere selected. The list of peptides was further reduced to 33 whenpeptides with undesirable motifs with possible toxic effects wereexcluded. A further seven peptides were excluded due to lack ofstructural alignment to HBD3. Of the remaining peptides, struc-tural modeling revealed that Cp-thionin-2 (KT43C), fabatin(LL44C), and mung bean defensin PDF-1 (KT46C) were mostclosely related structurally to HBD3 and displayed similar place-ment of hydrophobic and cationic residues (Fig. 1). Three otherpeptides, hevein-like AMP (QQ42G), antimicrobial seed protein(AC34H), and TK-AMP-D1 (R46C) (see Fig. S1 in the supple-mental material), share some structural features with HBD3, suchas the stacked antiparallel beta-sheets, and were carried forwardfor further analysis based on reported antibacterial activity. Addedto the list for analysis was the cyclic peptide circulin (C30P) (see

FIG 1 Structures of defensin peptides. Defensin-like peptides were modeledusing the Robetta protein structure prediction tool. Candidate peptide struc-tures were aligned to, and compared with, the empirically determined struc-ture of human beta-defensin 3 (HBD3; PDB ID 1KJ6) using the PyMOL Mo-lecular Graphics System. Peptides displaying structures most similar to HBD3are shown. The peptides are colored blue to red from the N to the C terminus.

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Fig. S1 in the supplemental material), which we define here asdefensin-like because it contains six cysteine residues that puta-tively form three disulfide bonds in the molecule. Two defensin-like peptides, gamma-2-purothionin (K47C) from wheat grainand Rs-AFP-1 (Q23N) from radishes, were initially excludedbased on the selection criteria, because they possess knottin mo-tifs. However, both peptides are structurally very similar to HBD3(see Fig. S1) (42) and were thus included for analysis. The anti-bacterial activities of the latter two peptides have not been char-acterized previously. The peptides are referred to below by theirIDs (i.e., identifier names generated from the amino acid se-quence [e.g., in “LL44C,” “LL” refers to the first two aminoacids, “44” to the length of the peptide, and “C” to the lastamino acid]) (Table 1).

In addition to including defensin-like peptides, we included inthe analysis a small linear heptapeptide (JC-Pep-7) from Jatrophacurcas and a synthetic tetrapeptide (O3TR) recently reported asdisplaying antimicrobial activity (43, 44) (Table 1). To modify thenet charges of the latter two peptides, an acetyl group was added tothe NH2 terminus during peptide synthesis. The peptides werechemically synthesized (Table 1), and their physicochemicalproperties were compared (Table 2). The net charges of the pep-tides ranged from �0.2 to 11.6; HBD3 was the most positivelycharged peptide. Isoelectric points ranged from 6.9 to 14, withmost in the range of 8 to 10.7. The majority of peptides displayednegative hydropathy scores (Table 2). The defensin-like peptidescontain 3 to 8 cysteine residues, while the small linear peptidescontain none.

Kinetics of action of AMPs. To identify AMPs showing anti-microbial activity in actively growing bacterial cultures, such asmight be encountered in food or beverage preparations, peptidesin the concentration range of 40 to 50 �g ml�1 were initially in-cubated with L. brevis in vitro for 1.5 h and were then furtherincubated in growth medium until control cultures, in the absenceof AMPs, reached stationary phase. Of the 14 AMPs tested, onlyHBD3, LL44C, KT43C, and O3TR showed antimicrobial activityagainst L. brevis under the chosen experimental conditions andconcentration range (Fig. 2) (see Materials and Methods). Thepeptides were subsequently tested for antimicrobial activityagainst a range of Gram-positive and -negative bacteria, chosenfor their importance as contaminants of food and beverages, as

well as several pathogenic bacterial species often associated withfood poisoning (Table 3). S. aureus was included as a positivecontrol, because this bacterium is regularly used for the assess-ment of AMP activity. By far, HBD3 showed the broadest hostrange under the chosen experimental conditions, displaying activ-ity against the Gram-positive bacteria L. brevis, P. dextrinicus, S.aureus, and B. cereus and against the Gram-negative bacterium P.aeruginosa but not against E. coli or Salmonella enterica. PeptidesLL44C and KT43C displayed activity against L. brevis, P. dextrini-cus, and P. aeruginosa but not against S. aureus, B. cereus, E. coli, orSalmonella enterica. The synthetic tetrapeptide O3TR displayedactivity only against L. brevis and P. dextrinicus, while an acetylatedform of the same peptide had no activity. The rest of the peptideswere ineffectual against the range of bacteria tested under the cho-sen experimental conditions. None of the peptides showed anti-microbial activity against the yeast Saccharomyces cerevisiae (datanot shown).

Peptides displaying antimicrobial activity were further testedat lower concentrations to determine the concentration requiredfor 95% growth inhibition after 48 h of incubation (IC95:48 h). For

TABLE 1 List of peptides

Peptide ID Source organism Amino acid sequenceSource orreference

Human beta-defensin 3 HBD3 Human GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK 33Cp-Thionin-2 KT43C Vigna unguiculata KTCMTKKEGWGRCLIDTTCAHSCRKYGYMGGKCQGITRRCYCLLNC 50Fabatin-2 LL44C Vicia faba LLGRCKVKSNRFNGPCLTDTHCSTVCRGEGYKGGDCHGLRRRCMCLC 51Mung bean defensin PDF-1 KT46C Vigna radiata KTCENLANTYRGPCFTTGSCDDHCKNKEHLRSGRCRDDFRCWCTRNC 52TK-AMP-D1.1 R46C Triticum kiharae RDCESDSHKFHGACFSDTNCANVCQTEGFAGKCVGVQRHCHCTKDC 53Hevein-like AMP QQ42G Euonymus europaeus QQCGRQAGNRRCANNLCCSQYGYCGRTNEYCCTSQGCQSQCRRCG 54Antimicrobial seed protein AC34H Phytolacca americana ACIKNGGRCVASGGPPYCCSNYCLQIAGQSYGVCKKH 55Circulin-A C30P Chassalia parviflora GIPCGESCVWIPCISAALGCSCKNKVCYRN 56Gamma-2-purothionin K47C Triticum aestivum KVCRQRSAGFKGPCVSDKNCAQVCLQEGWGGGNCDGPFRRCKCIRQC 57Rs-AFP-1 Q23N Raphanus sativus QKLCERPSGTWSGVCGNNNACKN 58JC-Pep-7 KV5K Jatropha curcas KVFLGLK 59Acetylated JC-Pep-7 Ac-KV5K Jatropha curcas Acetyl-KVFLGLK This studySynthetic tetrapeptide O3TR Synthetic OOWW 44Acetylated synthetic tetrapeptide Ac-O3TR Synthetic Acetyl-OOWW This study

TABLE 2 Physicochemical properties of peptides

PeptideNo. ofcysteines

Netcharge

Isoelectricpoint

Avghydropathyscore

Human beta-defensin 3 6 11.6 10.72 �0.7Cp-thionin-2 8 7.5 9.07 �0.439Fabatin-2 8 6.7 8.87 �0.423Gamma-2-purothionin 8 6.5 8.82 �0.596Hevein-like AMP 10 5.3 8.3 �1.038Antimicrobial seed protein 6 4.7 8.72 �0.1Mung bean defensin PDF-1 8 3.7 8.12 �1.174JC-Pep-7 0 3 14 0.914Rs-AFP-1 3 2.8 8.88 �0.597Circulin-A 6 2.6 8.13 0.416Acetylated JC-Pep-7 0 2 14 0.914Synthetic tetrapeptide 0 1 14 0.19Acetylated synthetic

tetrapeptide0 0 7 0.19

TK-AMP-D1.1 8 �0.2 6.92 �0.613

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L. brevis grown in continuous culture for 48 h in the presence ofpeptides, the IC95:48 h of HBD3, LL44C, KT43C, or O3TR was 1.3,10.3, 12.7, or 14.4 �g ml�1, respectively, and the IC95:48 h againstP. dextrinicus was in the same range (Table 3).

To examine the kinetics of inhibition in greater detail, the pep-tides, in various concentrations, were incubated in vitro for 1.5 hwith L. brevis as a model bacterium. Following the incubation,the bacteria were immediately plated onto MRS agar without theadded extended growth period (48 h) in growth medium. Theconcentration of peptide required to obtain 50% or 95% inhibi-tion of growth (IC50:1.5 h or IC95:1.5 h, respectively) was deter-mined (Fig. 3). Following the 1.5-h in vitro incubation, the IC95:1.5 h for HBD3, LL44C, KT43C, or O3TR was determined to be2.5, 13.7, 21.4, or 60 �g ml�1, respectively, while the IC50:1.5 h was2.2, 7.3, 10.5, or 19.6 �g ml�1, respectively. Comparison of theIC95:1.5 h (Fig. 3) to the IC95:48 h (Table 3) suggests that inhibi-tion is immediate for HBD3, LL44C, and KT43C, while for O3TR,prolonged incubation is required to achieve the same effect as theother peptides. We explored this apparent discrepancy betweenIC95:1.5-h and IC95:48-h values for O3TR by examining the sur-vival kinetics of L. brevis following incubation with O3TR at thetwo peptide concentrations (Fig. 4A). At the higher concentrationof peptide (IC95:1.5 h; 60 �g ml�1), inhibition was almost instan-

taneous, with the percentage of survival decreasing to 50% in 1min. Survival dropped to 28% after 10 min and to 7% by 60 min.At the lower concentration of peptide (IC95:48 h, 14.4 �g ml�1),survival dropped to 60% after 10 min and remained at this levelwhen the incubation period was extended to 60 min. Thus, atboth peptide concentrations, the survival kinetics appear to bebiphasic, with an initial rapid decrease in cell survival to ap-proximately 60% by 10 min, followed by a lower rate of killingthereafter (Fig. 4B).

With these facts in mind, the antimicrobial activities of the fourpeptides were directly compared at the IC95:1.5 h (Fig. 5). As ob-served in Fig. 4A, O3TR at the IC95:1.5 h resulted in an instanta-neous 53% reduction in survival, while 50% reduction wasachieved after 7 min with HBD3. Both KT43C and LL44C dis-played similar kinetics of inhibition, achieving 50% inhibition by40 and 44 min, respectively.

Thermostability and salt tolerance of peptides. To be effec-tive as antimicrobials in food and beverages, it may be advanta-geous in some cases (for example, bread making) for the peptidesto be thermostable. To examine this, peptides at the IC95:1.5 hwere incubated at 100°C for 10 min and were then cooled at 25°Cprior to addition to L. brevis cultures (Fig. 6A). The peptide-bac-terium mixture was then incubated for 1.5 h prior to being plated

FIG 2 Antimicrobial activities of AMPs in actively growing bacterial cultures.L. brevis (2 � 103 to 4.5 � 103 cells ml�1) in 50 �l PBS was incubated with 50�l of peptide in the concentration range of 40 to 50 �g ml�1 for 1.5 h in a96-well microtiter plate. One hundred microliters of 2� medium was thenadded, and cultures were incubated at 30°C for 48 h. Bacterial survival wasassessed by measuring the absorbance at a � of 540 nm. L. brevis culturesincubated in the absence of peptides served as the control (open bar). Errorbars represent the standard errors of the means from three independent ex-periments. Each experiment was conducted with triplicate samples.

TABLE 3 IC95 of peptides

Bacterium Gram stain

IC95 (�g ml�1)a

HBD3 LL44C KT43C O3TR AcO3TR C30P KT46C K47C R46C Q23N KV5K AcKV5K QQ42G AC34H

L. brevis Positive 1.3 10.3 12.7 14.4 40 50 40 50 50 50 50 50 40 45P. dextrinicus Positive 2.1 11.2 10.7 17.3 40 50 40 50 50 50 50 50 40 45S. aureus Positive 12.5 45 40 40 40 50 40 50 50 50 50 50 40 45B. cereus Positive 5.8 45 40 40 40 ND 40 ND ND ND 50 50 40 45S. enterica Negative 50 45 40 40 40 50 40 50 50 50 50 50 40 45E. coli Negative 50 45 40 40 40 ND 40 ND ND ND 50 50 40 45P. aeruginosa Negative 3.5 10.3 21.4 40 40 ND 40 ND ND ND 50 50 40 45a ND, not determined.

FIG 3 Inhibitory activities of HBD3, KT43C, LL44C, and O3TR. Cultures ofL. brevis (4.5 � 103 cells ml�1; 100 �l) were mixed with increasing concentra-tions of peptides (100 �l). Following incubation for 1.5 h at 30°C, 100 �l ofeach sample was added to MRS agar and was incubated for 48 h at 30°C.Bacterial survival was expressed as a percentage of the survival of untreatedcontrol cultures, taken as 100%. Error bars represent the standard errors of themeans from two independent experiments. Each experiment was conductedwith triplicate samples. Symbols: �, HBD3; �, KT43C; Œ, LL44C; �, O3TR.Lines were generated by nonlinear regression analysis of the data. R2 valueswere 0.9781 (�), 0.9836 (�), 0.9026 (Œ), and 0.9172 (�).

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for enumeration. The LL44C and O3TR peptides were thermo-stable, while KT43C was slightly inhibited (approximately 18%)by the pretreatment. However, the antimicrobial activity of HBD3was severely affected by the pretreatment, showing a 70% reduc-tion. The method of cooling after the heat treatment did not alterthe activities of the peptide, as evidenced by the fact that rapidcooling by plunging the mixture into an ice bath generated similarresults (data not shown).

To determine if the peptide activities are influenced by the saltconcentration, the AMP assay was repeated in PB containing dif-ferent concentrations of NaCl and using the IC50:1.5 h or lower forthe peptides (Fig. 6B). The antimicrobial activities of KT43C,LL44C, and O3TR were extremely sensitive to salt concentrations.Complete inhibition was achieved by KT43C and LL44C at NaClconcentrations of �100 mM and by O3TR at �50 mM. On theother hand, the activity of HBD3 was less affected by decreasingthe salt concentration: there was a slight decrease (approximately20%) in activity at NaCl concentrations of �50 mM. The same

trends in salt sensitivity for all four peptides were observed whenthe AMP concentrations were varied (data not shown).

Antimicrobial activity using peptide combinations. To de-termine if the peptides interact with each other to exert additive or

FIG 4 Comparison of antimicrobial activities of two concentrations of O3TR. (A) O3TR at the IC95:1.5 h (60 �g ml�1) (�) or at the IC95:48 h (14.4 �gml�1) (�) in 100 �l was incubated with 100 �l of L. brevis (4.5 � 103 cells ml�1) for increasing time intervals (0 to 60 min). Following incubation for 1.5 h at30°C, 100 �l of each sample was added to MRS agar and was incubated for 48 h at 30°C. Bacterial survival was expressed as a percentage of the survival of untreatedcontrol cultures, taken as 100%. Error bars represent the standard errors of the means from 3 to 5 independent experiments. Each experiment was conducted withtriplicate samples. (B) The data presented in panel A were normalized relative to the percentage of bacterial survival at 0 min (1.0). Error bars represent thestandard errors of the means.

FIG 5 Kinetics of action of the antimicrobial peptides. One hundred micro-liters of a peptide (HBD3 [�], KT43C [Œ], LL44C [�], or O3TR [�]) at theIC95:1.5 h was incubated with 100 �l of L. brevis (4.5 � 103 cells ml�1) forincreasing times (0 to 90 min). Following incubation, 100 �l of each samplewas added to MRS agar and was incubated for 48 h at 30°C. Bacterial survivalwas calculated as a percentage of the survival of the untreated bacterial sample.Error bars represent the standard errors of the means for three independentexperiments, with each experiment conducted with triplicate samples.

FIG 6 Thermostability and salt sensitivity of antimicrobial peptides. (A)The antimicrobial peptide HBD3, KT43C, LL44C, or O3TR (100 �l) at theIC95:1.5 h was heated to 100°C for 10 min (�) and then cooled to roomtemperature before addition to 100 �l of L. brevis (4.5 � 103 cells ml�1). (B)The peptide HBD3 (1.5 �g ml�1) (�), KT43C (10.5 �g ml�1) (�), LL44C (5.5�g ml�1) (Œ), or O3TR (14.4 �g ml�1) (�) and L. brevis (4.5 � 103 cells ml�1)were diluted in PB containing different NaCl concentrations. Following incu-bation for 1.5 h at 30°C, 100 �l of each sample was added to MRS agar and wasincubated for 48 h at 30°C. The percentage of inhibition (A) or of survival (B)was calculated relative to the level for untreated bacterial cultures, taken as100%. Error bars represent the standard errors of the means for three inde-pendent experiments, with each experiment conducted with triplicate sam-ples. **, P 0.001; *, P 0.05. For the percentage of survival (B), peptidesshowed values significantly different from the HBD3 values (for LL44C andO3TR, P 0.001; for KT43C, P 0.05) at �100 mM NaCl.

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reductive effects on their individual antimicrobial activities, L.brevis was incubated with each peptide individually or in combi-nation for increasing time intervals. Based on the kinetics of actionof each peptide (Fig. 5), a suboptimal concentration of each pep-tide, sufficient to assay its activity, was chosen in order to allow thequantification of combined effects. Accordingly, combinatorialexperiments were carried out with HBD3 and O3TR at concen-trations lower than the IC50:1.5 h (1.5 and 14.4 �g ml�1, respec-tively) due to their rapid kinetics, while LL44C and KT43C weremaintained at the IC95:1.5 h (13.6 and 21.4 �g ml�1, respectively),due to their slow kinetics of action (Fig. 5). HBD3 in combinationwith LL44C or KT43C resulted in a more rapid reduction in the

survival of L. brevis than that achieved by either peptide alone (Fig.7A and B). The increased rate of inhibition from the combinationof the two peptides was statistically significant between 10 and 50min for HBD3 and LL44C and between 5 and 50 min for HBD3and KT43C. Combining LL44C and KT43C also resulted in anincreased rate of inhibition (Fig. 7C). Combining the tetrapeptideO3TR with any of the three defensin peptides did not appear toincrease the rate of inhibition (Fig. 7D to F), except for the com-bination of O3TR with KT43C, where an immediate reduction insurvival at 1 min was observed (Fig. 7E). Thereafter, the rate ofinhibition for the combined peptides was not statistically signifi-cant from that for either peptide alone.

FIG 7 Interactions between antimicrobial peptides. Cultures of L. brevis (4.5 � 103 cells ml�1; 100 �l) were incubated for increasing times with the antimicrobialpeptides (100 �l), either singly or in pairwise combinations (Œ), at concentrations of 1.5 �g ml�1 for HBD3 (�), 13.7 �g ml�1 for LL44C (�), 21.4 �g ml�1 forKT43C (}), and 14.4 �g ml�1 for O3TR (Œ). The combination of peptides in each experiment is shown in the key on the right of each panel. Followingincubation for 1.5 h at 30°C, 100 �l of each sample was added to MRS agar and was incubated for 48 h at 30°C. Bacterial survival was calculated as a percentageof the survival of the control without AMP treatment (taken as 100%). Error bars represent the standard deviations from one to five independent experiments.Each experiment was conducted with triplicate samples.

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To determine if the increased rate of inhibition observed withsome of the combined peptides resulted from the peptides actingin synergy or in an additive manner, a combination index (CI)score was calculated using IC50:1.5 h to IC90:1.5 h as the measuredeffect (Table 4). Synergistic effects were observed using the pep-tide combinations HBD3 plus LL44C, HBD3 plus KT43C, andO3TR plus KT43C, although in the last case, synergism was evi-dent only in the first 10 min of the reaction (Fig. 7E). PeptideLL44C in combination with either KT43C or O3TR produced anadditive effect, while the combination of HBD3 and O3TR wasantagonistic. Taking the findings together, the most potent effecton bacterial inhibition was observed for the combination of HBD3with either of the plant defensin-like peptides KT43C and LL44C.

DISCUSSION

The discovery and characterization of a vast array of small pep-tides displaying antimicrobial activity offer great potential for thedevelopment of novel therapeutics to combat food poisoning andspoilage caused by pathogenic and nonpathogenic bacteria, espe-cially in light of the growing upsurge in resistance to conventionalantibiotics. Our previous studies had identified the peptide hu-man beta-defensin 3 (HBD3) as a potent inhibitor of a wide rangeof beer-spoiling bacteria, and expression and secretion of HBD3by lager yeast provided prophylaxis against bacterial infection inyeast fermentations (33). Since the bacterial species implicated inbeer spoilage are also associated with the spoilage of dairy-basedfood and other yeast-based fermented products, HBD3 is an ex-cellent candidate for development as a food additive to preventspoilage.

Here we set out to identify HBD3-like peptides and focusedon peptides of plant origin due to the diverse array of AMPspreviously identified in plants (27). Antimicrobial peptides inplants are generally stored in seeds as endospermal reserves,and humans and animals consume substantial quantities ofsuch seeds without any known ill effects. While more than 350defensin-like peptides have been described (41), to date theantimicrobial activity and host range of each peptide havemainly been studied individually using differing experimentalconditions. This has led a variety of reported IC and often-contradictory reporting on host ranges.

We developed a screening pipeline to select candidate peptidesfor a comparative analysis with HBD3, employing criteria such assource of peptides, elimination of peptides containing potentiallytoxic motifs, structural similarity to HBD3, and prior reportedantimicrobial activity. Peptides containing knottin motifs wereexcluded because of their similarity to scorpion toxin peptides,which have been shown to be cytotoxic to mammalian cells and

therefore would not be likely candidates for development as po-tential food additives. Two exceptions were made to the lattercriterion: peptides gamma-2-purothionin and RS-AFP-1 were in-cluded in the analysis due to their structural similarity to HBD3.By use of defined experimental conditions, the peptides weretested in parallel for antimicrobial activity against representativeGram-positive and Gram-negative food-spoiling bacteria orpathogenic bacteria associated with food poisoning.

Of the defensin-like peptides analyzed, only two, fabatin-2(LL44C) from the broad bean, Vicia faba, and Cp-thionin(KT43C) from the cowpea, Vigna unguiculata, displayed antimi-crobial activity; however, the IC95 was higher than that of HBD3.Both peptides were effective against L. brevis and Pediococcus dex-trinicus (recently renamed Lactobacillus dextrinicus) but notagainst S. aureus or B. cereus, which were inhibited by HBD3.HBD3, KT43C, and LL44C were ineffective against the Gram-negative species E. coli and Salmonella enterica; however, all threedisplayed antimicrobial activity against P. aeruginosa.

The structural analysis of the peptides identified fabatin-2, Cp-thionin-2, and mung bean PDF-1 as most structurally similar toHBD3; however, we found that mung bean PDF-1 did not showantimicrobial activity under the experimental conditions chosenhere. Likewise, the other peptides identified through structuralsimilarity to HBD3 did not display antibacterial activity. A com-parison of physicochemical properties of the peptides indicatedthat the only parameter that appeared to correlate with antimicro-bial activity was overall net charge, since HBD3, Cp-thionin-2,and fabatin-2 were the top three ranking peptides based on netpositive charge, while mung bean PDF-1 had a much lower netpositive charge. Thus, including net charge as a selection criterionmay be informative in identifying the most active peptides.

An analysis of the kinetics of the antimicrobial activity of thethree defensin-like peptides indicated that HBD3 has a muchhigher rate of activity than either KT43C or LL44C. However, theantimicrobial activities of KT43C and LL44C were greatly influ-enced by salt concentrations: at NaCl concentrations of 100mM, both peptides were more effective than HBD3. Previousstudies have shown that the antimicrobial activity of HBD3 wasnot influenced by salt concentrations (19, 45, 46); however, thesalt sensitivities of fabatin-2 and Cp-thionin-2 had not been ana-lyzed previously. Additionally, both peptides were resistant to heattreatment, while HBD3 was sensitive, a property that might beuseful if peptides are to be used in thermally processed foods.Thus, the plant peptides fabatin-2 and Cp-thionin-2 are good can-didates for the prevention of food spoilage (47). Given the ob-served salt sensitivities of fabatin-2 and Cp-thionin-2, it is possiblethat antimicrobial activity might be detected for the other nonac-tive peptides (Table 3) by reducing the salt concentrations in theassay. Likewise it will be interesting to determine whether the hostrange can be expanded at lower salt concentrations.

We also included in the analysis one nondefensin plant anti-microbial peptide, JC-Pep-7, from Jatropha curcas (Barbadosnut), and a synthetic peptide, named O3TR. Both peptides aresmall and easy to synthesize and would be more cost-effective thanthe longer defensin-like peptides, which range from 45 to 54amino acids. Of these nondefensin peptides, only O3TR displayedantimicrobial activity. This peptide was originally identified aspart of a rational design study to generate small cationic peptideswith antimicrobial potential (43). The tetrapeptide contains twoornithine residues, which are nonnatural charged amino acids

TABLE 4 Combination index scores of peptides

IC

CI scorea

HBD3 �LL44C

HBD3 �KT43C

KT43C �LL44C

HBD3 �O3TR

KT43C �O3TR

LL44C �O3TR

90% 0.47 0.34 ND 11.36 0.00 0.9980% 0.46 0.30 0.99 5.64 0.00 1.1270% 0.45 0.38 1.09 4.27 0.00 1.2060% 0.45 0.40 1.12 3.57 0.39 1.2550% 0.43 0.44 1.19 ND ND NDa ND, no data.

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with high resistance to proteases, and two tryptophans, whichhave a strong preference for the membrane interface. Subsequentmodifications of the peptide by modification of the N terminuswith p-hydroxyl-cinnamic acid or acetic anhydride improved theantimicrobial activity of the tetrapeptide. Further modifications,such as the addition of a fatty acyl tail to the N terminus, improvedthe antimicrobial activity, although the reported MICs and mini-mum bactericidal concentrations were higher than that observedfor unmodified O3TR in this study (44). In the present study, theacetylated form of the peptide was ineffectual against the range ofbacteria tested. The concentration of O3TR required to achieve a95% reduction in the bacterial load in 1.5 h of incubation wasapproximately 40-fold higher than the corresponding concentra-tion of HBD3 and approximately 3- to 5-fold higher than those ofLL44C and KT43C. Interestingly, O3TR displayed biphasic kinet-ics of antimicrobial activity. Upon addition to L. brevis, the pep-tide caused a rapid reduction in bacterial survival: there was analmost instantaneous 50% reduction of the bacterial load. There-after, the rate of bacterial killing was reduced. The initial high rateof killing was independent of the peptide concentration.

We examined the question of whether the peptides mightact in concert to improve antimicrobial activity by determiningthe rate of bacterial killing achieved by peptides individually orin pairwise combinations. Our analysis revealed that the defen-sin-like plant peptides KT43C and LL44C act in synergy withHBD3 to improve the rate of bacterial killing; on the otherhand, when combined with each other, the two plant peptidesproduced an additive effect. In combination with HBD3, thetetrapeptide O3TR was antagonistic, while it was additive withLL44C. There appear to be some synergistic effects for O3TR incombination with KT43C; however, synergism is apparent onlyimmediately upon incubation of the peptides with L. brevis (0 to10 min) and not thereafter.

Defensins are purported to exert antimicrobial activitythrough insertion into the plasma membranes of bacterial cells,leading to depolarization of the membrane and cell lysis (13, 48).Membrane insertion requires a critical minimum concentration.The very narrow concentration range required to reduce bacterialsurvival from 100% to 5% supports this mode of action. Theinteraction of defensins with the cell membrane is influenced byphysiological conditions, such as localized pH (46). There is alsoevidence indicating that HBD3 treatment leads to disorganizationof membrane-bound multienzyme machines, such as the cell wallbiosynthesis complex and the electron transport chain, even in theabsence of cell depolarization (19). The synergistic effects of de-fensin-like peptides may result from facilitation of the insertion ofone defensin-like peptide into the cell membrane by another,through the formation of membrane pores with mixed types ofpeptides or by physiological modulation of the pH or ionicstrength of the local environment. Further research will be re-quired to tease out the synergistic mechanism.

The mode of action of the ultrasmall cationic peptides, such asO3TR, is not well understood; however, recent evidence suggeststhat small cationic peptides can be inserted into phospholipid bi-layers, leading to disruption of essential processes, such as respi-ration and cell wall biosynthesis (49). Thus, similar cellular pro-cesses are disrupted by defensin and nondefensin cationicpeptides. The reason for the antagonistic effect of O3TR on HBD3activity is not understood, but this effect may point to an initial

competition for binding of the peptides to the bacterial mem-brane.

Taken together, the data presented indicate that the plantdefensin-like peptides fabatin-2 and Cp-thionin-2 are effectiveantimicrobials and may act in concert with HBD3 to controlfood-spoiling and food-poisoning bacteria. Comparative anal-ysis of the tertiary structures of the plant peptides and HBD3may aid in the design of synthetic peptides with enhanced an-timicrobial activity.

ACKNOWLEDGMENTS

This research was funded by a grant (13/F/462) from the Department ofAgriculture, Food and the Marine to U.B. The funders had no role in studydesign, data collection and interpretation, or the decision to submit thework for publication.

FUNDING INFORMATIONThis work, including the efforts of Ursula Bond, Michael C. Beckett, andJoanna Kraszewska, was funded by Department of Agriculture, Food andthe Marine (13/F/462).

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