eukaryotic antimicrobial peptides

8/12/2019 Eukaryotic Antimicrobial Peptides

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R : C o n c i s e R e v i e w s i n F o o d S c i e n c eJFS R: Concise Reviews/Hypotheses in Food Science

Eukaryotic Antimicrobial Peptides:Promises and Premises in Food SafetyTALI RYDLO, JOSEPH MILTZ, ANDAMRAM MOR

ABSTRACT: There is a lack of efficient and safe preservatives in the food industry. Massive use of some commonfood preservation methods has led, over the years, to development of a resistance to different treatments by variousfood pathogens. Enteric bacteria are especially tolerant to adverse environmental conditionssuch as low pH andhigh salt concentrations which limits efficiency of some preservation methods. Consumers demand for natural,preservative-free, and minimally processed foods and worldwide concern regarding disease outbreaks caused byfood-related pathogens have created a need for development of new classes of antimicrobial (AM) agents. The twen-tieth century revealed a massive array of new peptide-based antimicrobials. Small ribosomally made compoundsare found in practically alllivingspecies where they actas important component of host defense. Certain indubitableadvantages of peptidespertaining to simplicity, activity spectra, and bacterialresistanceover knownpreservativeagentsadvocatetheir potential for foodpreservation. Nisin, an AM compound originating from bacteria, is so far theonly FDA-approved peptide. However, a growing number of reportsdescribe the potential of animal-derived antimi-crobial peptides as food preservatives. These studies have yielded various native compounds and/or derivatives that

possessmarkedlyimprovedantimicrobialpropertiesunderabroadrangeofincubationconditions.Thepresentworkreviews the most investigated peptides and accounts for their potential use as alternatives to the preservatives usedtoday. Thefocusis on research aspects aimingat understanding themechanism of actionof these peptidesat extremeenvironments of various food systems. Collectively, the data accumulated are convincingly indicative of potentialapplications of these peptides in food safety, namely, with respect to fighting multidrug-resistant pathogens.

Keywords: antimicrobial packaging, antimicrobial resistance, drug design, food pathogens

Introduction

The use, and sometimes misuse, of antimicrobials in both hu-man and veterinary medicine during the years has resulted intheemergence of bacterialstrainsthat no longerrespondto antimi-

crobial therapy. So far, resistance has developed to almost all an-timicrobial drugs (Gold and Moellering 1996). Moreover, bacterial

strainsresistantto all availableantimicrobial agents havebeen iden-

tified among clinical isolates of different bacterial species (Tomasz

1994). A study aiming at evaluating the resistance of 5 major food-

borne pathogens (Campylobacter, Salmonella, Yersinia enterocol-

itica, pathogenicEscherichia coli, andListeria monocytogenes) iso-

latedfrom922meatproductsshowedthatentericbacteriaarehighly

resistant against a wide range of antimicrobial agents and also pos-

sess multiresistance phenotypes of distinct advantages (Mayrhofer

andothers2004). Thealarmingemergence of resistance among bac-

teria has led the WorldHealthOrganization( WHO)to announcean-

timicrobial drug resistance as a main public health concern (WHO

1995). Over the years, massive use of common food safety barri-ers led to the development of resistance by various food microor-

ganisms (Samson and others 1995; Holyoak and others 1996; Lin

and others 1996; Park and others 1996; Henriques and others 1997;

Kathariou 2002).

The difficulty in the food industry in preventing outbreaks of

foodborne pathogens such asSalmonella, E. coliO157:H7,Staphy-

lococcus, L. monocytogenes, and Clostridium perfringensresulted in

MS 20060146 Submitted 3/2/2006, Accepted 8/26/2006. Authors are withDept. of Biotechnology & Food Engineering, TechnionIsrael Inst. ofTechnology, Haifa, 32000, Israel. Direct inquiries to author Mor (E-mail:[email protected]).

thousands deaths andmillioncases of foodborneillnesseseach year

(Mead and others 1999). In addition to well-known pathogens that

may cause global pandemics, new unrecognized and uncontrolled

pathogens are emerging as a result of changing ecology and tech-

nology or by the transfer of mobile virulence factors such as bac-teriophage, thus dramatically changing the spectrum of foodborne

illnesses(Tauxe 2002). Enteric bacteria are especially tolerant to ad-

verse environmental conditions such as low pH and high salt con-

centrations (Small and others 1994;Cheville and others 1996;Brown

and others 1997; Mayrhofer and others 2004). Virulent strains ofE.

coliareincreasingly recognizedas foodbornepathogens.Amongthe

6 virotypes, enterohemorrhagicE.coli(EHEC) are considered to be

highly significant due to their low infectious dose and the severe

consequences of infection (Buchanan and Doyle 1997). Escherichia

coliO157:H7 may cause hemorrhagic colitis and hemolytic uremic

syndrome (Riley and others 1983). Outbreaks of foodborne illness

due to E. coliO157:H7 have been reported from various parts of the

world (Doyle 1991; Griffin and Tauxe 1991; Besser and others 1993;Anonymous 1995). Acid foods such as apple juice were reported to

be associated with these outbreaks (Steele and others 1982). There-

fore, contamination and proliferation of food pathogens are a great

concern for food safety and public health.

Consumer awareness and demands as well as food legislation

have made the task of providing high-quality products even more

challenging to the food industry. Consumers demand higher qual-

ity, preservative-free, safe yet mildly processed foods with an ex-

tended shelf life. Since acidity and sterilization are the most com-

mon preservation techniques that control outgrowth of pathogenic

spore-forming bacteria, addressing this consumers need calls for

innovative approaches to ensure product preservation.

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Promises and premises in food safety . . .

Antimicrobial Peptides

Antimicrobial peptides (AMPs) are widely distributed in na-ture and are used by nearlyif notall life forms as essentialcomponents of nonspecific host defense systems. The list of dis-

covered AMPs has been constantly increasing, mostly in the last

2 decades (representative AMPs are shown in Table 1). The skin of

frogs (Csordas and Mich 1970) and lymph of insects (Habermann

1972) were initially shown to contain peptides that kill bacteria in

culture. Since then, more than 800 AMPs were described in many

living organisms: microorganisms, insects,amphibians, plants, andmammals (Boman 1995; Nicolasand Mor1995; Hancockand Lehrer

1998; Simmaco and others 1998; Lehrer and Ganz 1999; Tossi and

others 2000; Cleveland and others 2001; Mor 2001). Moreover, typi-

cal mammalianpeptides may be produced in fungi as well (Mygind

and others 2005).

In addition to their ability to kill microorganisms directly, AMPs

seem to be able to recruit andpromotevarious elements of host im-

munity (Boman 1995; Gudmundsson and Agerberth 1999; Hancock

and Diamond 2000; Scott and Hancock 2000). AMPs are produced

bothconstitutively and by induction, predominantlyin the animals

most exposed tissues (for example, skin, eyes, andlungs), which are

mostlikelytocomeincontactwithmicroorganisms.Theyarerapidly

synthesized at low metabolic cost, easily stored in large amounts,and have a wide spectrum as well as synergistic activity, thus pro-

viding the producing organism with a broad spectrum of coverage

against a wide range of pathogens (Bals 2000).

Structure-activity relationship (SAR) studies of various AMPs

demonstrated that the molecular size of native peptides can be sig-

nificantly reduced while maintaining antimicrobial properties and

sometimesimproving them (Mor andNicolas 1994b; Oh andothers

1999; Chen and others 2000; Feder and others 2000; Tossi and oth-

ers 2000; Kustanovich and others 2002; Gaidukov and others 2003;

Fazioand others2005;Radzishevski and others2005; Robinsonand

others 2005;Shalev and others 2005; Rydlo and others 2006). Similar

optimization attempts were reported also using nonnative model

peptide sequences (Blondelle and Houghten 1992; Haynie and oth-

ers 1995; Park and others 2004). There are a growing number of

reports using biotechnology techniques either for SAR studies or

for mass production of AMPs (Kilara and Panyam 2003; Chen and

others 2005; Donini and others 2005; Zho and others 2005).

Table 1 --- Amino acid sequence of representative gene-encoded antimicrobial peptides

Designation Sequencea Reference

AMPs produced by microbial cellsNisin ITSISLCTPGCKTGALMGCNMKTATCHCSIHVSK Rogers 1928Pediocin PA1 KYYGNGVTCGKHSCSVDWGKATTCIINNGAMAWATGGHQGNHKC Nielsen and others 1990Leucocin A KYYGNGVHCTKSGCSVNWGEAFSAGVHRLANGGNGFW Hastings and others 1991Sakacin P KYYGNGVHCGKhSGCTVDWGTAIGNIGNNAAANWATGGNAGWNK Tichaczek and others 1994Bacteriocin 31 ATYYGNGLYCNKQKCWVDWNKASREIGKIIVNGWVQHGPWAPR Tomita and others 1996

Enterocin A TTHSGKYYGNGVYCTKNKCTVDWAKATTCIAGMSIGGFLGGAIPGQC Aymerich and others 1996Enterocin P ATRSYGNGVYCNNSKCWVNWGEAKENIAGIVISGWASGLAGMGH Cintas and others 1997

AMPs produced by animal cellsMagainin GIGKFLHSAKKFGKAFVGEIMNS Zasloff 1987MSI-78 GIGKFLKKAKKFGKAFVKILKKCONH2 Ge and others 1999aPR-39 RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFP Shi and others 1996Spheniscin SFGLCRLRRGFCAHGRCRFPSIPIGRCSRFVQCCRRVW Thouzeau and others 2003Pleurocidin GWGSFFKKAAHVGKHVGKAALHTYL Cole and others 1997Dermaseptin S4 ALWMTLLKKVLKAAAKALNAVLVGANA Mor and Nicolas 1994aK4S4(1-14) ALWKTLLKKVLKAACONH2 Rydlo and others 2006Cecropin P1 SWLSKTAKKLENSAKKRISEGIAIAIQGGPR Lee and others 1997Melittin GIGAVLKVLTTGLPALISWIKRKRQQ Wilcox and Eisenberg 1992LL-37 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES Johansson and others 1998Clavanin A VFQFLGKIIHHVGNFVHGFSHVF Lee and others 1997Curvacin A ARSYGNGVYCNNKKCNVNRGEATQSIIGGMISGWASGLAGM Ganzle and others 1999

aAmino acid sequence in the 1 letter code. Positively charged residues are highlighted in bold and underlined.

Biosynthesis, Structural Features,and Mechanism of Action

Animal-derived AMPs are synthesized as primary translationproducts, prepropeptides, which are further processed to givethe active forms (Boman 1995; Nicolas and Mor 1995). In addition

to proteolytic processing, posttranslation modifications include, in

somecases, glycosylation(Bulet and others 1993), carboxy-terminal

amidation, amino acid isomerization (Simmaco and others 1998),

halogenation (Shinnar and others 1996), and cyclization (Tang and

others 1999). Some AMPs can be derived from proteolysis of func-tionalpolypeptides such as skin PYY(sPYY) (Vouldoukis andothers

1996), buforin II (Kim and others 2000), and lactoferricin (Ulvatne

and Vorland 2001) or from proteins a priori not suspected to be

involved in antimicrobial function (Rotem and others 2006).

AMPs are often produced as closely related multimembered fam-

ilies that may vary in only a few amino acid residues (Nicolas and

Mor 1995). Within each family there is a remarkably high conserva-

tion of cDNA and amino acid sequence in the prepro regions, sug-

gesting their functional significance, although comparison of pep-

tides from all organisms, even those that are closely related, shows

practically no conservation of amino acid sequence. Despite the

enormous variety of sequences and structures, AMPs possess cer-

tain common features (Boman1995; Nicolas and Mor 1995; Andreuand Rivas 1998; Hancock and Lehrer 1998). They are usually made

of less than 50 amino acids, bear a net positive charge due to an

excess of basic (often lysine and/or arginine) over acidic residues,

and contain about 50% hydrophobic amino acids. They often fold

into 3-dimensional amphipathic structures stabilized by cysteine

disulphide bridges. Linear peptides lacking cysteines tend to fold,

only upon contact with membranes, into a variety of amphipathic

helixes,pleated-sheets, loops, or lessdefined extended structures in

which positively charged hydrophilic domains are well delineated

from hydrophobic domains. These features seem to be the main

factors affecting their known and diverse biological activities.

The mechanismof actionof AMPs seems to involve multiple tar-

gets. The most cited target is the plasma membrane while more re-

cent studies suggest intracellular targets at least for some peptides

(Zasloff 2002; Brogden 2005). Whether representing a final or inter-

mediate step in themechanism of action, it is clear that theinterac-

tion of AMPs with theplasma membrane plays an importantrole in

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their biological activity (Figure 1). Accordingly, cytotoxicity results

from nonspecific interactions involving either membrane pertur-

bations (Matsuzaki 1998; Wu and others 1999; Huang 2000) and/or

cytoplasmic translocation followed by interaction with anionic el-

ements such as nucleic acids (Friedrich and others 2000; Brogden

2005). In the case of cell-wall containing microorganisms (for ex-

ample, Gram-negative bacteria), the external membranes may act

asadditionalbarriers.IthasbeenproposedthatAMPsfirsttargetthe

external membrane and undergo a self-promoted uptake (Hancock

1997), a process that involves displacement of divalent cations fromtheir bindingsite on lipopolysaccharides (LPS)in theexternalmem-

brane by competitor peptides. This leads to increased permeability

and access to the cytoplasmic membrane. Hydrophobic peptides,

which often aggregate in aqueous media, fail in this competition,

resulting in poor bactericidal potency. Due to their nonspecific tar-

get and mode of action, the generation of resistance toward these

antimicrobial agents was shown to be less likely to occur (Navon

and others 2002; Yeaman and Yount 2003).

Although most AMPs act by nonspecific mechanisms, they of-

ten display some selectivity between different microorganisms, for

example, Gram-negative compared with Gram-positive bacteria

(Boman and others 1991;Meister and others 1997), fungi preference

over other eukaryotic cells (Tailor and others 1997), as well as nor-mal compared with cancerous mammalian cells (Utsugi and others

Figure 1 --- Proposed antibacterial mechanism of action oflinear AMPs: Unfolded cationic peptides associate withthe negatively charged surface of the outer membrane(OM) and neutralize the charge over a patch of the mem-brane or competitively displace divalent cations fromtheir binding sites on lipoplysacharides (LPS), creatingcracks through which peptides can cross the outer mem-brane. In the periplasmic space (PS), the peptides ad-here to negatively charged phospholipids in the cytoplas-mic membrane (CM), which induces their amphipathicfold. Insertion of multiple monomers within the mem-brane lipid core ultimately leads to disruption of the mem-brane structure and function (Hancock 2001; Rydlo andothers 2006).

1991; Perez-Paya and others 1994). The basis for this discrimina-

tion appears to be linked to a number of parameters, including lipid

composition, membranefluidity, extent of trans-membrane electric

potential (Andreu and Rivas 1998; Matsuzaki 1999; Tossi and others

2000), and peptides self-assembly in solution (Pouny and others

1992; Ghosh and others 1997; Feder and others 2000; Kustanovitch

and others 2002; Radzishevski and others 2005; Rydlo and others

2006).

Potential Therapeutic Applications

Many AMPs exhibit potent activity against a wide range of mi-crobes, including most known Gram-negative and Gram-positive bacteria, yeasts, and fungi, but also against enveloped

viruses such as HIV, herpes simplex virus, influenza A virus, and

vesicular stomatitis virus (Zhangand Hancock 2000; Lorin and oth-

ers 2005). AMPs also display activity against eukaryotic parasites,

including trypanosomes, malaria parasites, and nematodes(Ghosh

and others 1997; Krugliak and others 2000; Dagan and others 2002;

Efronand others2002) anda variety of types of tumor cells (Kamysz

andothers 2003; Papoand others 2004). Most AMPs display minimal

inhibition concentration (MIC) at low micromolar range, including

against antibiotic-resistant microorganisms. Many AMPs kill bacte-

ria faster than conventional bactericidal antibiotics and are not af-fected by antibiotic-resistance mechanisms that often limit the use

of other antibiotics. For example, no evidence of cross-resistance

between magainin and antibiotics has been documented in clinical

use (Ge and others 1999a). The peptidesnatural role(s) may thus

involve synergy both with each other, as seen with dermaseptins

(Mor and others 1994a), and with other agents of the host defense

system. Magainin shows synergistic killing with another skin AMP

termed PGLa (Matsuzaki and others 1998). Synergy has also been

shown with lysozyme and various antibiotics against selected wild-

type and mutant bacteria (Yan and Hancock 2001), with antifungal

or antiprotozoan agentsas well as with theanticancer drug doxoru-

bicin, when tested against fungi, protozoa, and cancer cells, respec-

tively (Scott and others 1999).

AMPs are currently studied intensively because of their advan-

tages over conventional antibiotics. They show prospects to be

usedin various antimicrobialapplications: Microbe-derived antibi-

otic peptides (for example, Gramicidin S and Polymyxin B) have

been used, over the years, mostly as topical creams and solutions

(Hancockand Chapple1999).Currently, severalclinical trialsare on-

going to assess various topical treatments by animal-derived AMPs

(Hancock and Patrzykat 2002). These include phase III trials of IB-

367 for treating oral mucositis, most commonly associated with ra-

diotherapy or chemotherapy for cancer; phase II clinical trials of

IB-367 in aerosol formulation for Pseudomonas aeruginosa lung in-

fections in cystic fibrosis. An indolicidin, MBI-226, is undergoing

phase III clinical trials for sterilization of insertion sites for central

venous catheters, and other indolicidin-like peptides are being in-vestigated in phase II clinical trials for therapy of acute acne.

Additional potential applications are also being considered, in-

cluding (1) therapy of stomach ulcers due to Helicobacter pylori

infections (Projan and Blackburn 1993); (2) contraceptive agents

limiting the spread of sexually transmitted diseases from Neisse-

ria, Chlamydia, human immunodeficiency virus, and Herpes sim-

plexvirus (Yasin and others 2000); (3) imaging probes for bacterial

and fungal infections (Welling and others 2000; Melendez-Alafort

and others 2004); (4) agents enhancing the potency of existing

antibiotics, by facilitating access of antibiotics into the bacterial

cell (Darveau and others 1991); (6) introduction of antimicrobial

genes into plants, which by expressing the peptide become resis-

tant to pathogens (Osusky and others 2000); (7) delivery agents for

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conjugated drugs and compounds (Feder and others 2001; Futaki

andothers2001;Hariton-Gazalandothers2002);and(8)foodsafety.

The food-related potential applications are detailed below.

Food Preservation

Consumers concern about possible adverse health effects ofsome food additives has stimulated research of new effectivealternatives. The bacteriocins produced by Gram-positive LAB bac-

teria have been found to be appropriate candidates to fulfill these

requirements, mostly due to their natural origin in foods such asfermented dairy and meat products and due to their safety aspects

(Cleveland and others 2001; Chen and Hoover 2003; Papagianni

2003). Nisin, a bacteriocin produced byLactococcus lactisbacteria,

was found to be of a particular interest. Discovered in 1928 (Rogers

1928), nisin wasinitially evaluatedas a clinicalantibioticin the1940s

(Hirsch andMattick 1949).Later, it wasfoundto be suitable forfood

preservation due to its lethal activity against foodborne pathogens

and spoilage microorganisms, inhibition of cell wall biosynthesis

and spore outgrowth, and, most of all, its safe use and consump-

tion with no apparent adverse effects (Hurst 1981; Montville and

others 1995). So far, nisin is the only purified antimicrobial pep-

tide approved by the U.S Food and Drug Administration for use in

particular food products (FDA Federal Register 1988). It is used in acrude extract containing up to 5%of nisin of the solid. Of the differ-

ent variants of nisin, the most used commercial form is NisaplinTM,

which contains 2.5% of the active ingredient (Nisin A), 77.5% NaCl,

and 12% nonfat dry milk (Chen and Hoover 2003). Currently it is

permitted for use mostly in dairy products (especially cheese) and

canned goods (vegetables, soups). In European countries it is also

used in baby foods, baked goods, mayonnaise, and milk shakes.

Nisin however, displays several shortcomings: low solubility at

physiologic pH reduces its activity and limits its use in most cured

meat products (Rayman and others 1983), and it is inactive against

yeast, molds, and Gram-negative bacteria, unless other processing

technologies are used in combination. These technologies include

adding chelator agents likeEDTA (Cutter and Siragusa1995, Branen

and Davidson 2004), preheating the product (Boziaris and others

1998), or pH reduction (Rayman and others 1983). The partial suc-

cess of nisin as a food preservative has prompted examination of

other bacteriocins. Pediocin PA-1 showed the most promising re-

sults (Nielsen and others 1990). But it is not yet an approved food

additive in theUnitedStates. Theuse of bacteriocinsin food preser-

vation presentsseriouslimitations because of their relatively narrow

activity spectra and moderate antibacterial effects.

Synthetic peptides have been tested in food products such as

apple juice. SAR studies of synthetic model AMPs aiming at un-

derstanding their mechanism of action resulted in a 14-residue,

long peptide, 8K6L, composed of 8 lysine and 6 leucine residues

(Blondelle and Houghten 1992; Haynie and others 1995). The pep-

tide showed bactericidal effect against E. coli O157, reducing itspopulation by 6 log units after 1 h of incubation at the concen-

tration of 50 g/mL in a buffer medium (Appendini and Hotchkiss

1999). A more recent study aimed at using this synthetic peptide as

a preservative in food packaging materials demonstrated the ability

of thepeptide to reduce E.coliO157 population by 3.5log unitsafter

10-min incubation in citrate buffer (pH 3.5) at a concentration of

5 g/mL (Appendini and Hotchkiss 2000). Testing the bactericidal

effectof thepeptidein apple juice at 25 C (pH3.7) revealedonly 3.5

log unit reduction after a long incubation period of 8 h at the high

peptide concentration of 100 g/mL.

As pointed out above, unlike lactobacteria AMPs that inhibit

growthof mainly Gram-positive organisms, AMPs thatare produced

by animal cells often display activity against a much larger spec-

trum of microorganisms. The potential of several native AMPs as

food preservatives is increasingly being reported. The main results

from typical studies are summarized below according to the pep-

tides origin.

Mammalian AMPsA 26 amino acid peptide derived from the sequence of PR-

39, a proline-arginine rich polypeptide isolated from porcine neu-

trophils, was found to be effective in killingE. coli, Salmonella Ty-

phimurium, andStreptococcus suis, at 37 C (Shi and others 1996).The peptide was proposed to penetrate the outer membrane ofE.

coli, gain entry into thecytoplasm,and thus affect bacterialviability

by interfering with DNA and/or protein synthesis (Boman and oth-

ers 1993; Cabiaux and others 1994). Scanning electron microscopy

studies of bacterial cells exposed to PR-26 indicated that the pep-

tidedoes notlyse cells by pore-formingmechanisms (Shiand others

1996). Its effectiveness against pathogenic strains E. coliO157:H7

andL. monocytogeneswas tested at different temperatures (Anna-

malai and others 2001). Although after 24-h incubation the peptide

decreased bacterial populations by 4 and 5 log units at 24 and 37C, respectively, it had poor activity at the lower temperatures rep-

resenting normal refrigeration.

AvianAMPsTwo beta-defensins, termed spheniscins, were recently isolated

from the stomach content of the king penguinAptenodytes patago-

nicus(Thouzeauandothers2003).Ithasbeenproposedthatincom-

bination with other AMPs, spheniscins may be involved in a long-

termpreservationoffoodinthemalebird sstomach.This38-residue

AMP displayed mainly bacteriostatic effect against Gram-negative

bacteria but showed bactericidal effect against most Gram-positive

bacteria testedat a concentration range of 0.4to 15M and was ac-

tive against yeast and filamentous fungi at concentrations ranging

from 1.5 to >100 M. The peptide showed stable activity at a range

of moderate acidity (pH 4 to 6), suggesting that it is not affected by

conserving conditions in the stomach. No studies aiming to char-

acterize these peptides in food systems as preservatives have been

published to date.

FishAMPsProtamine, extracted from fish milt, demonstrated antimicrobial

activity against a range of Gram-negative and Gram-positive bac-

teria, yeasts, and molds (Islam and others 1984; Uyttendaele and

Debevere 1994; Johansenand others1995). This 30 amino acid AMP

(of which 66% are arginine) is believed to disrupt the cytoplasmatic

membrane by inducing leakage of K+, ATP, and intracellular en-

zymes of sensitive cells (Islam and others 1987; Johansen and oth-

ers 1997; Stumpe and Bakker 1997). In recent studies this AMP was

evaluated for its efficacy againstL. monocytogenesandE. coliat pH

levels ranging from 5.5 to 8 (Hansen and Gill 2000) and at temper-atures ranging from 5 to 30 C (Hansen and others 2001). The pep-

tideshowedbetteractivityagainstGram-negative bacteria,showing

similar MIC and MBC values, but was mainly bacteriostatic toward

Gram-positivebacteria.Inbothcasesthepeptidesactivityincreased

at alkaline pH in a medium containing positively charged protein

(gelatin A) where the competitive electrostaticinteractions between

protamineand culturemediaand bacterialsurface were in favor for

thelatter. Additionof 0.9mM EDTA wasfoundto be eithersynergis-

tic to protamine (reduced MIC) or bactericidal, depending on the

bacterialstraintested.The effect of temperatureon protamine MICs

varied greatly among strains,species, and genera. Therefore, no def-

inite conclusion regarding the effect of temperature on bactericidal

potency could be made.



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Another fish AMP, pleurocidin, isolated from the edible win-

ter flounder, was active against Gram-positive and Gram-negative

bacteria andwas heat andsalt tolerant (Cole andothers 1997, 2000).

Pleurocidin was active against foodborne microorganisms at levels

well below the legal limit for nisin (10,000IU/g) without significant

effect on human red blood cells (Burrowes and others 2004), indi-

cating its potentialas a foodpreservativeand a natural alternative to

conventional chemicals. It is noteworthy, however, that pleurocidin

was inhibited by magnesium and calcium (Cole and others 2000),

which may limit the use of this AMP in environments laden withthese cations.

AmphibianAMPsInadditionto mammalian-like neuropeptidesandhormones, the

skin of amphibians also contains a rich arsenal of broad-spectrum,

cytolytic AMPs (Nicolas and Mor 1995; Simmaco and others 1998).

Prepro-dermaseptins are processed and then stored in the large

granules of dermal glands (Nicolas and others 2003) and released

onto the skin surface upon stimulation to provide an effective and

fast-acting defense against noxious microorganisms (Bevins and

Zasloff 1990; Simmaco and 1998). All known amphibian AMPs are

linear and able to form amphipathic helical structures in hydropho-

bic environments (Papagianni 2003). As wide-spectrum microbi-cides, amphibian AMPs stimulate increasing interest because of

their unique characteristics and potential therapeutic usefulness.

Two of the most investigated representative frog AMPs, magainins

and dermaseptins, are discussed below.

Magainins discovery was triggered by the observation that

wounded frogs manage to thrive in waters dense with bacteria

(Zasloff 1987).Initially, themagainins were isolated from theskin of

the African clawed frogXenopus laevis, but have also been found in

their stomach (Moore and others 1991). These 21 to 27 residueAMPs

form an amphipathic -helix upon binding to membranes (Marion

and others 1988) and displayed a broad spectrum of antimicrobial

activity. At micromolar concentrations, they induced lysis of bacte-

ria,fungi,protozoa,and tumor cells (Zasloffand others 1988; Soballe

and others 1995). SAR studies yielded a 22-residue peptide termed

MSI-78 showing improved potency and broader spectrum of activ-

ity, including against multiresistant bacterial species (Ge and others

1999a), and was found to be suitable for development as human

therapeutic agent. A formulation based on this peptide has been

taken through all phases of clinical trials for topical treatment of

infecteddiabeticfoot ulcers (Ge and others 1999b). The bactericidal

effect of magaininand itssynthetic analogs was investigated against

13 food-related pathogens (Abler and others 1995) displaying MICs

ranging from


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Effectsof temperatureOnly limited bactericidal activity of AMPs was reported at low

temperatures(4and20 C)whenexaminingtheinactivationoffood-

borne pathogens with magainin analogs (Alber and others 1995).

Low incubation temperatures may affect the peptides solubility,

leading to formation of activity-limiting aggregates. Maisnier-Patin

and others (1996) found that the antimicrobial effect of the bac-

teriocin EFS2 on L. innouua was highest at 35 C, with activity

loss at 15 C due to low solubility. But this may not be the case

for other peptides: Annamalali and others (2001) reported thatactivity of PR-26 against E. coli O157:H7 and L. monocytogenes

was reduced at 4 and 10 C compared to 25 and 37 C. Similarly,

Mendoza and others (1999) reported that as temperature is de-

creased (from 37 to 6 C) the bactericidal effect of enterocin AS-48,

which is produced byEnterococcus faecalisand tested onL. mono-

cytogenes, decreased substantially. Both of the above-mentioned

peptides were highly soluble at low temperatures, indicating that

low temperatures may alter susceptibility to antimicrobial agents

rather by affecting fluidity and/or fatty acid profile of bacterial cell

membrane.

For other peptides, lower temperatures have an opposite effect

due to structure stabilization. The C-terminal domain of class IIa

bacteriocins, a region involved in receptor recognition, was used tostudy the effects of temperature on structure and antimicrobial ac-

tivity (Kaur and others 2004). Peptides that did not possess a 2nd

C-terminal disulfide bond in addition to the N-terminal disulfide

bond were found to experience partial disruption of the helical sec-

tion at elevated temperatures (37 C) and were 30- to 50-fold less

potent. In a study on dermaseptins (Rydlo and others 2006), low

temperatures of 25C and 4 C were found to limit the bactericidal

activity of dermaseptin S4 derivatives (Figure 2). The difference in

potency could not be linked to obvious changes in peptides sec-

ondary structure. Moreover, the peptide exhibited the most ordered

structure at 25 C although it was most active at 42 C. A slight de-

crease in the bacterial population at 4 C in the control group (that

Figure 2 --- Effects of incubation conditions on the antimicrobial properties of dermaseptin derivatives on E. coli0157:H7. Plotted in column A are data obtained after 2 h of incubation with a 28 residue dermaseptin. ColumnsB and C present data obtained with a 14-residue truncated derivative and its acylated version, respectively, at samepeptide concentration (8 M). CFU values represent the mean standard deviations (SD) obtained from two inde-pendent experiments performed in duplicates. Bars represent the SD from the mean. Absence of a bar indicates thatthe SD value is smaller than the symbol size. Zero CFU indicate negative cultures (Rydlo and others 2006).

was not exposed to the peptides) suggests that bacterial resistance

increased due to stress conditions.

Effects of saltElectrostatic interactions between peptide and negatively

charged targets maybe masked at high salt concentrations. Salt

might also affect peptide organization in solution in terms of either

secondarystructure, salting outor induce aggregation,thus limiting

its activity. The-helical content of a synthetic bactericidal peptide

[RLLR]5 was reduced from 72% to 13% in the presence of 200 mMNaCl, leading to an 8- to 32-fold decreasein its antimicrobial activity

against bacteria and fungi (Park and others 2004). This study also

indicated that conjugation of -helix-capping motives at the pep-

tides termini helped maintain its helical structure under salty con-

ditions, resulting in improved activity. In different studies, however,

salt supplement actually improved bactericidal activity: Adding up

to 300 mM NaCl did not impair cecropin P1 activity againstE. coli

but progressively led to activity loss for magainin (Lee and others

1997). Activity of nisin againstE.coliO157 was achieved at high salt

concentrations of 5% irrespective of whether bacteria were precul-

tured in the presence or absence of salt in the incubation medium

(Ganzle and others 1999). Salt resistance to -helical cationic AMPs

was also reported againstP. aeruginosaPAO1 at salt concentrationsof 300 mM (Freidrich and others 1999). Unfortunately, none of these

studies addressed changes in secondary structure in the presence

of high salt concentrations.

The bee venom melittin was reported to convert from a

monomeric random coil to a helical tetramer as ionic strength was

increased from 0 to 500 mM NaCl (Wilcox and Eisenberg 1992).

Furthermore, 2 -helix forming peptides produced by gene engi-

neering methods were able to maintain the secondary structure at

extremely high salt concentrations up to 1.5 M (Kojima and others

1996). High salt concentration was explained to mask electrostatic

repulsion between similarly charged side chains of lysine (in acidic

conditions)orglutamicacid(inalkaliconditions)onthehydrophilic



7/11


surface of the helix bundle, thus stabilizing the peptide structure. In

addition, hydrophobic interaction was strengthened when increas-

ing salt concentration, thus facilitating peptide association.

Another study showed that the human cathelicidin, LL-37, con-

vertedfromrandomcoilinwaterto -helixinthepresenceofvarious

salts,including160mMNaCl,andthatthe-helixcontentcorrelated

theobservedantibacterialactivity (Johanssonand others 1998). The

salting out effect explained in this study causes peptide oligomer-

izationobservedwith increasing helicalcontent. Reducingrepulsive

forces between positively charged residues along the peptide chainwas also explained as an outcome of salt addition, resulting in sta-

bilization of the helix structure.

Changes in antibacterial activity of dermaseptin derivatives did

not correlate with structural changes as function of salt concen-

tration since the peptides exhibited the highest helical structure at

6% NaCl where activity was significantly reduced (Rydlo and others

2006). Acylationremarkablyimprovedefficacy, enablingthe peptide

to eliminate large bacterial populations (107 CFU) at high salt con-

centrationsof1.5%(approximately0.3MNaCl)asshowninFigure2.

At 3%, on the other hand, the peptide did not display improved ac-

tivity. At such high salt concentrations, growth of the control group

was also hampered.

Effects of pHVariations in pH can affect the peptides net charge, which in turn

affects its secondary structure, binding properties, and cytotoxic

activity. Histidine-containingamphipathichelical peptides werere-

portedtoexhibitrandomcoilstructureatacidicpHlevelsandtypical

-helix structure at basicconditions (Vogt and Bechinger 1999). An-

otherhistidine-basedpeptide,clavaninA,showedenhancedactivity

at acidicconditions while at neutral pH, the uncharged peptide be-

came inactive. Its substituted analog, clavanin AK, having a higher

pKavalue, exhibitedpotencyat all conditions (Leeand others 1997).

The human cathelicidin LL-37 displayed random coil structure at

acidicconditions (pH2 and3.8) and-helixat neutral andbasicpH

levels (Johansson and others 1998). The acidic conditions report-

edly destabilizedion pairs between acidicand basic side chainsdue

to the protonated state of acidic residues, whereas at basic condi-

tions, repulsive forces between basic side chains were limited due

to deprotonation of positively charged residues, leading to helix sta-

bilization. Thus, a peptides pKa value can determine loss or gain of

activity as a function of the environmental pH conditions.

Dermaseptin S4 derivatives also displayed reduced bacterici-

dal potency againstE. coliO157:H7 at acidic conditions of pH 3.6

(Figure 2). A few possible reasons might account for differences in

peptideefficacy: Bacterialstressresponsesmay reducebacterialsus-

ceptibilityto AMPs eitherby modificationsof themembranecompo-

sition,SOS geneexpression, or modificationof the trans-membrane

potential. Several studies revealed that rpoS, a gene involved in reg-

ulating theexpressionof a variety of stressresponse genes,could beinduced in response to a number of inimical processes used in the

food industry, including changes in water activity, osmotic shock,

and highly acidic conditions (Foster and Spector 1995; Fang and

others 1996; Turner and others 2000; Dodd and Aldsworth 2002).

Preculture ofE. coli in acidic conditions led to resistance to cur-

vacin A (Ganzle and others 1999). A similar outcome was obtained

with dermaseptins where bacteria that had been pre-incubated in

pH 3.6 and assayed in neutral pH became less susceptible (Rydlo

and others 2006). However,the fact that bacterial viability was ham-

pered in absence of peptide may point to stress responseadaptation

of bacteria being the cause for reduced potency.

As part of the stress response, it has been reported that E. coli

transmembrane potential decreases significantly at acidic condi-

tion (Richard and Foster 2004). The pH effect is bound to influence

the peptides interaction with bacterial membrane components in

a variety of manners. In support of this view, surface plasmon reso-

nance (SPR) experiments performed with dermaseptin derivatives

revealedhighbinding affinity at low pH buta low tendency to pene-

trateintothe membraneinnercore (Rydlo andothers2006). A solid-

stateNMR spectroscopy study also reported histidine-rich peptides

to alter their orientation in a model membrane as a function of the

surroundingpH, changingfrom parallelto transmembrane orienta-

tions, respectively, at acidic and elevated pH conditions (Bechinger1996).The pH differences also affected themode of actionby which

clavanin A permeabilized Lactobacillus sakemembrane (van Kan

and others 2002). Whereas the peptide efficiently released fluo-

rophores from unilamellar vesicles at neutral pH according to a

nonspecific permeabilization mechanism, it did not permeabilize

model bilayers at low pH levels although displaying antimicrobial

activity at those conditions.It was therefore suggestedthat thispep-

tide uses distinct modes of action at acidic and neutral pH.

Bactericidal Activity in Food Model Systems

Avariety of studies attempted to define the efficacy of severalpeptides in food model systems such as apple juice, milk, andmeat products: Activity of magainin, pediocin PA-1, and sakacin Awas decreased in the presence of bovine serum albumin or foods of

highproteincontent (Muriana1993; Abler and others 1995). Activity

of dermaseptin S4 derivatives in apple juice was limited by 2-fold

for certain derivatives (Yaron and others 2003) and up to 8-fold for

other derivatives (Rydlo and others 2006) compared with standard

incubation conditions. After preincubation in apple juice, the pep-

tide was assayed for its activity in standard LB medium showing

reduced activity comparedto nonpreincubated peptide. Thus, pep-

tide inactivation also depends on juice component(s). Activity of

nisin againstL. monocytogenesin fluid milk decreased as the milk

fat content increased (Jung and others 1992). Therefore, it has been

speculated that components such as lipids, proteins, and sugars in

foods interact with the peptide and hamper its activity.

Food Packaging

Antimicrobial packaging has become one of the most interest-ing and challenging topics in the area of active packaging (AP).In AP, the product, the package, and the environment interact dur-

ing food preparation and storage, resulting either in an improved

product quality and safety and an extended shelf life or in the at-

tainment of some product characteristics that cannot be obtained

by any other means (Miltz and others 1995; Yam and others 2005).

The most developed AP technology is oxygen scavenging. A reduc-

tion of oxygen in a package can inhibit oxidative reactions as well

as the growth of microorganisms. However, a reduction in the oxy-

gen concentration in a package to very low levels may encourage

the growth of pathogenic anaerobic microorganisms. Antimicrobialpackaging overcomes this problem. Several antimicrobial packag-

ing systems have been proposed and reviewed in detail (Appendini

and Hotchkiss 2002) where most of them use synthetic additives.

The subject of antimicrobial packaging has been reviewed recently

in 2 additional articles by Suppakul and others (2003a, 2003b). In

recent years, however, the public perception is that synthetic agents

may cause side effects and therefore consumers prefer natural over

synthetic additives. Miltz and others (2004) have developed an an-

timicrobial filmcontainingthe natural components of basil: linalool

and methyl chavicol. This film has shown a significant inhibition of

E. coliand Listeriaas well as other microorganisms and extended

the shelf life of cheddar cheese. Miltz and others (2006) have stud-

ied very recently the properties of an antimicrobial coating based



8/11

R

C

i

R

i

i

F

dS

i


on a dermaseptin AMP. Thiscoating has shown significant microbial

growth inhibition.

Concluding Remarks

Whilethe data collectedin this review highlight part ofthe mas-sive research that is currently being conducted, many signsdesignate the potential of natural AMPs in food preservation. Fu-

ture challenges lie in our ability to adapt these extraordinary com-

pounds to perform tasks specifically related to food safety. Better

understanding of themode(s) by which AMPs rapidly eliminate mi-

croorganismsshouldprovide solid groundsfor engineering newand

upgradedderivativeswithoptimizedpotency andstability under the

range of incubation conditions typical to food. This endeavor is in-

deed stimulating an increasing number of multidisciplinary studies

that tackle these issues by a large variety of strategies: So far, suc-

cess in significantly reducing the molecular size while increasing

resistance to proteases was achieved through SAR studies aiming

at optimizing known native peptides or model peptide sequences

through conventionaland/or combinatorial methods.In the future,

the use of biotechnology techniques should further enable mass

production of AMPs to minimize their costs. The recent isolation

of plectasin, a defensin-like AMP expressed in fungi, clearly repre-

sents a breakthrough toward achieving several of these goals. Thereis therefore little doubt that as progress is made, new derivatives

will be designed which will enhance the suitability of these simple

yet phenomenal molecules for various antimicrobial applications,

including food safety, for their demonstrable ability to escape most

of the known resistance mechanisms.

AcknowledgmentsThe authors gratefully acknowledge the financial support by THE

ISRAEL SCIENCE FOUNDATION (grant nr 387/03) and by funds

from the Technions Vice President for Research.

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