Recent Patents on DNA & Gene Sequences 2008, 2, 000-000 1

1872-2156/08 $100.00+.00 © 2008 Bentham Science Publishers Ltd.

Biomolecules as Host Defense Weapons Against Microbial Pathogens

Marco Dalla Rizza1,*

, Paola Díaz Dellavalle

1, Rafael Narancio

1, Andrea Cabrera

1 and Fernando

Ferreira2

1Biotechnology Unit, National Institute of Agricultural Research, INIA Las Brujas, Ruta 48 Km 10, Canelones,

Uruguay, 2Faculty of Chemistry, University of Uruguay (UdelaR), Montevideo, Uruguay

Received: April 21, 2008; Accepted: April 29, 2008; Revised: April 29, 2008

Abstract: Antimicrobial peptides have been considered a new source of biomolecules in several fields of

research/innovative applications: they would adjust to an ideal behavior seeking to overcome clinicians, microbiological,

human-animal-plant-environmental concerns. Antimicrobial peptides can be considered as ancient weapons found in

living organisms suggesting they have played a fundamental role in his successful co-evolution with pathogens. Acting on

microorganism membrane or having intracellular targets, they can also act as effectors of the innate immune response

resulting on non-specific mechanisms of action. Two elements have speeded the research on pathogen control alternatives:

a verified increase of antibiotic resistance and the relevance of finding amenable environmental compounds in plant

health. As a result of its importance, great efforts have been accomplished to find, characterize, combine and synthesize

effective antimicrobial peptides. This review intends to emphasize the generation of biomolecules, whether native or

synthetic analogues, that have been matter of recent patents. Developments of biomolecules suitable for therapeutic

scopes and agricultural use have several challenges such as intrinsic toxicity, in vivo stability and suitable formulation

contemplating the cost of production. Thus, biotechnological procedures using microbial systems or transgenic crop as

plant factories might help to solve these challenges.

Keywords: Antimicrobial, peptides, modes of action, biocontrol agents.

INTRODUCTION

It is widely accepted among professionals of several fields that antibiotic resistance will be promptly a global social concern in the fight against pathogen infections [1]. Two of the major contributors to these infections are widespread over prescription and misuse of antibiotic drugs, practices that have promoted the dissemination of a series of particularly harmful bacterial strains that resist to conventional antimicrobial treatments. Another potential main reason for the developed bacterial resistance in humans, is the massive use of preventive antibiotics in animal food. Because of a fast growth rate, the frequencies of genetic mutations and selections, and the ability of bacteria to rapidly exchange genes, bacterial resistance to antibiotics seems to take place swiftly in the evolution of the bacterial development. [2] On plants, several diseases caused by pathogens (viruses, bacteria and fungi) affect plant crops resulting in yield losses and decreasing the quality and safety of agricultural products. Their control relies on finding plant genetic resistance and/or chemical pesticides, currently subjected to restrictions and regulatory requirements. [3]

It is likely that many of the new antibiotics currently in development will not be approved by the US Food and Drug Administration. Most of these drugs in development are analogs of previous antibiotics which work on a select number of bacterial targets. [4] Consequently, the priority for the next decades should be focused in the development of alternative drugs and/or the recovery of natural molecules

*Address correspondence to this author at the Biotechnology Unit, National

Institute of Agricultural Research, INIA Las Brujas, Ruta 48 Km 10,

Canelones, Uruguay; Tel: +598 2 3677641; Fax: +598 2 3677609; E-mail: mdallarizza@lb.inia.org.uy

that would allow the consistent and proper control of pathogen-caused diseases. Ideally, these molecules should be as natural as possible, with a wide range of action over several pathogens, characterized by different ways of action, easy to produce, and not prone to induce resistance [1]. In addition to the natural peptides, thousands of synthetic variant peptides have been produced, which also share similar structures [5].

The new generation of native and synthetic peptide mole-cules, also known as AntiMicrobial Peptides (AMPs), isolated from a full range of organisms and species from bacteria to man, seem to fit this description. As a consequence, they have been named as ‘natural antibiotics’, because they are active against a large spectrum of microorganisms, including bacteria and filamentous fungi, in addition to protozoan and metazoan parasites. All of these molecules are key elements directly implicated in the innate immune response of their hosts, which includes the expression of fluid phase proteins that recognize pathogen-associated molecular patterns, instead of specific features of a given agent to promote their destruction. As a result, the response is very fast, highly efficient and applicable to a wide range of infective organisms. [1] In plant protection there is a need of new compounds in plant health that fit into the new regulations (compounds being more selective, with lower intrinsic toxicity and reduced environmental impact); in fact, several countries have banned many of them due to regulatory changes in pesticides registration requirements, generating difficulties on plant diseases control. Therefore, AMPs are being considered as candidates for plant protec-tion products. [3]

The knowledge acquired in the past two decades and the discovery of new groups of antimicrobial peptides, make

2 Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 Rizza et al.

natural antibiotics the basic element of a novel generation of drugs for the treatment of bacterial and fungal infections [1]. In addition, the wide spectrum of antimicrobial activities reported for these molecules suggests they potential benefit in the treatment of cancer and viral or parasitic infections. Different therapeutic applications of these compounds, from topical administration to systemic treatment of infections, have been developed by several biotechnological companies (http://www.inimexpharma.com; http://biotech.deep13. com/Alpha/alpha.html; http://www.geniconsciences.com/). [1]

Interestingly, to date, clinical Phase I and II trials have demonstrated a limited resistance for the bacterial strains tested. These features make the antibiotic peptides a power-ful arsenal of molecules that could be the antimicrobial drugs of the new century as an innovative response to the increa-sing problem of multi-drug resistance (MDR) (http://www. multi-drug-resistance.org; http://www.demegen.com). [1]

The antibiotics peptides are divided into two classes: the non-ribosomally synthetized peptides and the ribosomally synthetized (natural) peptides. The former are often drastically modified and are produced by bacteria, fungi, and streptomycetes. The latter instead, of wider distribution, are produced by all species of life (including bacteria) as a major component on the natural host defense molecules of these species. [6]

Antimicrobial peptides are evolutionary ancient weapons that can be found in organisms ranging from bacteria to plants, invertebrates and vertebrates species, including mammals. [7] Their widespread distribution throughout the animal and plant kingdoms, suggests that antimicrobial peptides have served a fundamental role in the successful evolution of complex multicellular organisms; being part of the ancient, nonspecific innate immune system, which is the principal defense system for the majority of living organisms [7,8].

These small and diverse peptides were initially isolated in the 1980s, from frogs and insects and in the former example, they have been found to play a vital role in their survival in bacteria infested swamps [9]. Since then, a large number of additional antimicrobial peptides has been found virtually everywhere in nature and it is difficult to categorize, except broadly on the basis of their net charge and/or their secondary structure [10]. Several hundred different antimicrobial peptides, from many different organisms, have been characterized to date; they are already listed in the publicly available databases including Swissprot and TrEMBL (http://www.expasy.org/sprot/sprot-top.html), RCSB Protein Databank (http://www.rcsb.org/pdb/home/ home.do), Antimicrobial Peptide Database (http://aps.unmc. edu/AP/main.html), Antimicrobial Sequences Database (http://www.bbcm.units.it/~tossi/antimic.html), and there are indications of the fact that this number will continue to grow rapidly. [2,11,12]

Collectively, the antimicrobial peptides display direct microbicidal activities toward Gram-positive and Gram-negative bacteria, fungi [13-19], some protozoan parasites [20] and viruses [21]. The importance of these activities in contributing to host defense may vary between different sites

within a particular organism and also between different types of organism. [7]

The expression of the antimicrobial peptides can be constitutive or can be inducible by ‘therapeutic’-inducing substance such as sodium butyrate [12], infectious and/or inflammatory stimuli, such as proinflammatory cytokines, bacteria, or bacterial molecules that induce innate immunity, e.g., lipopolysaccharides (LPS). In multicellular animals, they may be expressed systemically (for example, in insect haemolymph or vertebrate immune cells) and/or localized to specific cell or tissue types in the body most to infection, such as mucosal epithelia and the skin. [7,22]

DIVERSITY AND BIOLOGICAL ACTIVITY OF CATIONIC PEPTIDES

Cationic peptides is the largest group and the first to be reported; to date, hundreds of such peptides have been identified and over 50% of them have been isolated from insects. [1,7] They are typically relatively short (12 to 100 amino acids), and positively charged (net charge of +2 to +9) due to lysine and arginine residues and a substantial portion (around 50%) of hydrophobic residues. [6,23] They are found in all species of life, ranging from plants and insects to animals, including mollusc species, crustaceans, amphibians, birds, fish, mammals and humans [6,24]. The cationic pep-tides have a broad spectrum of antimicrobial activity including activity against both Gram-negative and Gram-positive bacteria, fungi, eukaryotic parasites and viruses. [4]

The fundamental structural principle of these peptides is the ability of the molecule to adopt a shape in which clusters of hydrophobic and cationic amino acids are spatially organized in discrete sectors of the molecule (termed ‘amphipathic’ design). [8] This feature allows the peptides to interact well with membranes that are composed of amphi-pathic molecules, especially negatively charged bacterial membranes. For the most part, animal cells tend to have membranes with no net charge so they are unaffected by cationic peptides. [4]

All cationic peptides are derived from larges precursors, including a signal sequence. Post-translational modifications include proteolytic processing, and in some cases glycosy-lation, carboxi-terminal amidation, amino-acid isomeri-zation, and halogenation. The diversity of sequences is such that the same peptide sequence is rarely recovered from two different species of animal, even those closely related. However, both within the antimicrobial peptides from a single species, and even between certain classes of different peptides from diverse species, significant conservation of amino acid sequences can be recognized in the preproregion of the precursor molecules; the design suggests that constraints exist on the sequences involved in the translation, secretion or intracellular trafficking of this class of membrane-disruptive peptide. [4,8]

The diversity of cationic peptides discovered is so great that it is difficult to categorize them except broadly on the basis of their secondary structure [25]. Basically, this peptides can be classified in four major classes: -helices, -sheet (peptides with two to four -strands stabilized by disulphide bonds), loop structures, and extended peptides [26], the first two classes being the most common in nature.

Biomolecules as Host Defense Weapons Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 3

The NMR solution structures of a list of some well-studied AMPs of the major classes are shown in Table 1.

FROM PROKARYOTES

Antimicrobial peptides produced by bacteria were among the first to be isolated and characterized [7]. While they do not offer protection against infection in the classical sense, they contribute to the survival of individual bacterial cells by killing other bacteria that might compete for nutrients in the same environment [7].

As shown in Table 2 [27-33] investigators have been patenting aminoacidic sequences of bacterial antimicrobial peptides, also called bacteriocins, produced by many or most bacteria. This AMPs are generally extremely potent compared with most of their eukaryotic counterparts, their activities may be either narrow or broad spectrum [7], capable of targeting bacteria within the same species or from different genera (see Table 2 US7238515).

The bacteriocins constitute a structurally diverse group of peptides and the classification into two broad categories has been recently proposed: lanthionine containing (lantibiotics) and non-lanthionine containing [7]. Lantibiotics are charac-terized by the inclusion of the unusual amino acid lanthionine and the necessity for postranslational processing to acquire their active forms. The most extensively studied lantibiotic is nisin, produced by Lactococcus lactis; which has been the center of attention because of their application as food preservatives without significant development of resistance. [1,7] A high number of lantibiotic aminoacidic sequences has been described and patented in the last years (see EP1169340 [34] and US0196900 [35]; these patents and others are referenced in Table 2).

FROM EUKARYOTES

PLANTS

In plants, it is widely believed that antimicrobial peptides play an important and fundamental role in defense against infection by bacteria and fungi [3,7,36]. Observations to support this role include the presence and expression of genes encoding antimicrobial peptides in a wide variety of plant species (see some relevant patents in Table 3 [37-50]). There are many demonstrations of their bactericidal and fungicidal activity in vitro, and correlations between expression levels of peptides and susceptibility to a given pathogen or the extent of resistance of a particular bacterium to plant-derived peptides and its virulence [7,36]. The two major and best-studied groups were thionins and defensins. The thionins with a molecular weight of 5 kDa, are generally basic and contain 6 or 8 conserved cysteine residues [51,52]. Physiologically relevant concentrations of thionins are active against bacteria and fungi in vitro, and studies utilizing transgenic plants have shown that heterologous expression of thionins can confer protection against bacterial challenge. The plant defensins, is a group of small AMPs (45-54 amino acids), highly basic cysteine-rich peptides that are apparently ubiquitous throughout the plant kingdom and display antibacterial and antifungal activities. To date, sequences of more than 80 different plant defensin genes from different plant species are available [36,53-56] and isolation of these has been recently patented (Table 3, US7238781,

US6911577, US6770750 and EP1849868). Consistent with a defensive role, they are particularly abundant in seeds, but have also been described in leaves, pods, tubers, fruit and floral tissues [17,57]. In the Defensins Knowledgebase (http://defensins.bii.a-star.edu.sg/) there are lists of patents referred to plant defensins (Table 3, e.g. WO009174 and US037100).

A new family of antimicrobial peptides has been described from Macadamia integrifolia and the first purified member has been termed MiAMP2c (Table 3, US7067624). The peptide, active against a number of plant pathogens in vitro, derives from a precursor protein similar to vicilins 7S globulin proteins, suspected of a putative participation in defense during seed germination [1].

INVERTEBRATES

Since invertebrates lack the adaptive immune system found in vertebrate species, they are reliant solely upon their innate immune systems to counteract invading pathogens. Considering the extraordinary evolutionary success of this group of organisms, it is evident that invertebrate innate immune mechanisms are extremely effective. [7] One component of the defense weapons, developed by inverteb-rates to rapidly eliminate invading pathogens, is the fast and massive production of potent AMPs [2]. To demonstrate the effectiveness of invertebrates antimicrobial peptides the researchers have isolated and evaluated in vitro and in vivo the AMPs; in Table 4 [58-68] are listed some patents related.

They are found in the haemolymph (plasma and hemocy-tes), in phagocytic cells, and in certain epithelial cells of invertebrates. They can be expressed constitutively, for example, in the hemocytes of marine arthropods such as shrimps, oysters, and horseshoe crabs (Table 4, US5861378), or induced in response to pathogen recognition, such as antifungal peptides in Drosophila. [7]

Although usually cationic, the primary structures of insects AMPs vary markedly. Members of the most frequent AMP families adopt an -helical conformation in memb-rane-mimetic environments [11]; these are the -helical cecropins. This is a family of 3 – 4 kDa linear peptides described in the haemolymph of insects in the early 1980s. Patenting of isolation and amino acid sequentiation of these peptides has also been done in 1982 (Table 4, US4355104). Cecropins were the first animal inducible AMPs to be isolated and fully characterized; these molecules are devoid of cysteine residues and contain two distinctive helical segments, a strongly basic N-terminal domain and a long hydrophobic C-terminal helix, linked by a short hinge. [1,2] The cecropins have a broad spectrum of activity [69] (see patent US5962410 [70]), some cecropins are capable of inhibiting cell-associated production of HIV-1 by suppres-sing HIV-1 gene expression [71]. Some C-terminus modifi-cations (cecropin-like peptides) exhibit increased potency and broader spectrum of antimicrobial activity than the cecropins, this was claimed in patent (Table 4, US5166321).

Another family, that is commonly found in insects is represented by peptides with high content in one or two particular amino acids, most frequently proline and/or glycine residues [2,72]. However, the most abundant group of antimicrobial peptides in invertebrates is the defensins,

4 Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 Rizza et al.

Table 1. Description of Some Antimicrobial Peptides Isolated from Different Sources, Selected as Representative Examples of their

Structural Class. High-Resolution Images of Peptide Backbones were Obtained from RCSB PDB

(http://www.rcsb.org/pdb/home/home.do) and PDBsum (http://www.ebi.ac.uk/)

Melittin

PDB code: 1bh1

Class: -helical

Source: Apis mellifera (Honeybee venom)

Amino acid sequence: GIGAVLKVLTTGLPALISWIKRKRQQ

Activity: Gram +, Gram -, Virus, Fungi, Mammalian cells, Cancer cells

Authors: Barnham, K.J., Hewish, D., Werkmeister, J., Curtain, C., Kirkpatrick, A., Bartone, N., Liu,

S.T., Norton, R., Rivett, D.

Ref.: [27]

Magainin 2

PDB code: 2mag

Class: -helical

Source: Xenopus laevis (Epithelial tissue of the African frog)

Amino acid sequence: GIGKFLHSAKKFGKAFVGEIMNS

Activity: Gram +, Gram -, Fungi, Cancer cells

Authors: Gesell, J.J., Zasloff, M., Opella, S.J.

Ref.: [28]

Lactoferricin B

PDB code: 1lfc

Class: -hairpin

Source: Bos taurus (Bovine)

Amino acid sequence: FKCRRWQWRMKKLGAPSITCVRRAF

Activity: Gram +, Gram -, Virus, Fungi, Cancer cells

Authors: Hwang, P.M., Zhou, N., Shan, X., Arrowsmith, C.H., Vogel, H.J.

Ref.: [29]

Human -defensin-1 (hBD-1)

PDB code: 1e4s

Class: -sheet

Source: Homo sapiens (Human, extracellular protein)

Amino acid sequence: DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK

Activity: Gram +, Gram -, Fungi, Virus, Chemotactic

Authors: Bauer, F., Schweimer, K., Kluver, E., Adermann, K., Forssmann, W.G., Roesch, P., Sticht, H.

Ref.: [30]

Antifungal protein 1 (RS-AFP1)

PDB code: 1ayj

Class: Alpha Beta

Source: Raphanus sativus var. Niger (Radish seeds)

Amino acid sequence:

EKLCERPSGTWSGVCGNNNACKNQCINLEKARHGSCNYVFPAHKCICYFPC

Activity: Gram +, Fungi

Authors: Fant, F., Borremans, F.A.M.

Ref.: [31]

Biomolecules as Host Defense Weapons Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 5

(Table 1) Contd….

Thanatin

PDB code: 8tfv

Class: Loop

Source: Podisus maculiventris (Insect, haemolymph tissue)

Amino acid sequence: GSKKPVPIIYCNRRTGKCQRM

Activity: Gram +, Gram -, Fungi

Authors: Mandard, N., Sodano, P., Labbe, H., Bonmatin, J.M., Bulet, P., Hetru, C., Ptak, M., Vovelle, F.

Ref.: [32]

Indolicidin

PDB code: 1g89

Class: Extended

Source: Synthetic construct based on Bos taurus sequence (Bovine neutrophils)

Amino acid sequence: ILPWKWPWWPWRR

Activity: Gram +, Gram -, Fungi, Virus

Authors: Rozek, A., Friedrich, C.L., Hancock, R.E.

Ref.: [33]

Table 2. Recent Patents Related to AMPs Isolated from Prokaryotes

Publication

number Title Inventors

Publication

date Source Ref. No.

US7238515 Anti-Listeria bacteriocin Berjeud, J.M., Fremaux, C., Cenatiempo,

Y., Simon, L 2007/07/03 Lactobacillus sakei [34]

US7166468

Production of the lantibiotic

cinnamycin with genes isolated

from Streptomyces cinnamoneus

Bibb, M.J., Widdick, D. 2007/01/23 Streptomyces

cinnamoneus [35]

US5594103 Lantibiotic similar to a nisin A De Vos, W.M., Roelant, J.S., Kuipers, O.P. 1997/01/14 Lactococcus lactis [36]

US7247306 Bacteria strain and bacteriocin

produced therefrom

Fliss, I., Desbiens, M., Lacroix, L., Tahiri,

I., Benech, R., Kheadr, E. 2007/07/24

Carnobacterium

divergens [37]

US6541607 Sublancin lantibiotic produced by

Bacillus subtilis 168 Hansen, N. 2003/01/04 Bacillus subtilis [38]

EP1908774

Antibacterial and antiviral

peptides from Actinomadura

namibiensis

The designation of the inventors has not

yet been filled (Not designed) 2008/04/09

Actinomadura

namibiensis [39]

US6989370 Bacteriocins and novel bacterial

strains

Stern, N.J., Svetoch, E.A., Urakov, N.N.,

Eruslanov, B.V., Volodina, L.I., Kovalev,

Y.N., Kudryavtseva, T.Y., Perelygin, V.V.,

Pokhilenko, D., Levchuk, V.P.,

Borzenkov, V.N., Svetoch, O.E., Mitsevich

E.V., Mitsevich I.P.

2008/01/22 Paenibacillus and

Bacillus species [40]

6 Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 Rizza et al.

Table 3. Selection of Relevant Patents Covering AMPs from Plants

Publication

number Title Inventors

Publication

date Source

Ref.

No.

US6150588 DNA encoding antimicrobial

proteins from Impatiens

Attenborough, S., Broekaert, W.F., Osborn,

R.W., Ray, J.A., Rees, S.B., Tailor, R.H. 2000/11/21 Impatiens balsamina [44]

US5750504 Antimicrobial proteins Broekaert, W.F., Cammue, B.P.A., Osborn,

R.W., Rees, S.B. 1998/05/12 Impatiens sp. [45]

US6187904 Biocidal proteins Broekaert, W.F., Cammue, B.P.A., Osborn,

R.W., Rees, Terras, F.R.G., Vanderleyden, J. 2001/02/13

Brassicaceae,

Compositae,

Leguminosae

members

[46]

US5905187 Antimicrobial peptides and plant

disease resistance based thereon

Duvick, J.P., Rood, T.A.,

Rao, A.G. 1995/05/18 Zea mays [47]

US7238781 Plant defensin polynucleotides

and methods thereof

Famodu, O.O., Herrmann, R.,

Lu, A.L., McCutchen, B.F.,

Miao, B.F., Miao, G.,

Presnail, J.K., Weng, Z.

2007/07/03

Dimorphotheca,

Picramnia,

Parthenium,

Nicotiana, Vernonia,

and Helianthus

[48]

US6909032

DNA encoding a Macadamia

integrifolia anti-microbial

protein, constructs comprising

the same and plant material

comprising the constructs

Manners, J.M., Marcus, J.P., Clifford, K.,

Green, J.L., Harrison, S.J. 2005/06/21

Macadamia

integrifolia [49]

US7067624 Antimicrobial proteins Manners, J.M., Marcus, J.P.,

Goulter, K.C., Green, J.L., Bower, N.I. 2006/06/27

Macadamia

integrifolia [50]

US6770750

Small and cystein rich antifungal

defensins and thionins like

proteins genes highly expressed

in the incompatible interaction

Oh, B.J., Ko, M.K., Shin, B. 2004/08/03 Arabidopsis thaliana [51]

WO009174

Antimicrobial protein and

peptide analogs of plant

defensins for pharmaceutical and

agricultural use

Posthuma, G.A., Schaaper, W.M., Sijtsma, L.,

Van Amerongen, A., Fant, F., Borremans, F.A. 2001/08/02 Raphanus sativus [52]

WO080032 Antifungal protein Reddy, V.S., Hassari, A.,

Isalm, A. 2006/08/03 Cicer arietimun [53]

US6864068 Antifungal proteins Rees, S.B., De Samblanx, G.W., Broekaert,

W.F. 2005/03/08 Raphunus sativus [54]

US6911577 Defensin polynucleotides and

methods of use

Simmons, C.R., Navarro Acevedo, P.A.,

Harvell, L.,

Cahoon, R., McCutchen, B.F.,

Lu, A.L., Herrmann, R.,

Wong, J.F.H.

2005/06/28

Zea mays, Oryza

sativa, Triticum

aeativum, Glycine

max, Beta vulgaria,

Hedera helix, Tulipa

fosteriana, Tulipa

gesneriana,

Cyamopsis

tetragonoloba

[55]

Biomolecules as Host Defense Weapons Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 7

(Table 3) Contd….

Publication

number Title Inventors

Publication

date Source

Ref.

No.

EP1849868 Plant defensins Weng, Z., Miao, G.,

Famodu, O.O. 2008/02/20

Dimorphotheca,

Picramnia,

Parthenium, and

Nicotiana

[56]

US6086885

Anti-bacterial protein extracts

from seeds of marigold and

paprika

Ziegenfus, S., Brinkhaus, F., Greaves, J. 2001/01/17 Tagetes,

Capsicum [57]

Table 4. Selection of Relevant Recent Patents Covering AMPs from Invertebrates

Publication

number Title Inventors

Publication

date Source

Ref.

No.

US6891085

Nucleic acid encoding the FUS6 antimicrobial

polypeptide of Agrotis Ipsilon and its use to

enhance disease resistance in a plant

Altier, D.J., Herrmann, R., Lu,

A.L., McCutchen, B.F., Presnail,

J.K., Weaver, J.L., Wong, J.F.H.

2005/05/10 Agrotis ipsilon

(Insect) [65]

US7202214 Rhizoc3 antimicrobial polypeptides and their

uses

Altier, D.J., Herrmann, R., Lu,

A.L., McCutchen, B.F., Presnail,

J.K., Weaver, J.L., Wong, J.F.H.

2007/04/10 Agrotis ipsilon

(Insect) [66]

US6063765 Antibacterial protein Benich, H., Axén, A., Carlsson,

A., Engström, A. 2000/05/16

Hyalophora

moths (Insect) [67]

US6331522 Antibacterial and antifungal peptide Bulet P, Hetru C, Hoffman R,

Sabatier L. 2001/12/18 Scorpion [68]

US6642203 Crustacean antimicrobial peptides Destoumieux, D., Bachere, E.,

Bulet, P. 2003/11/04

Penaeid prawns

(Crustacean) [69]

US4355104 Bacteriolytic proteins Hultmark, D., Steiner, H.,

Rasmuson, U., Boman, H.G. 1982/08/19

Hyalophora

cecropia

(Insect)

[70]

US5861378 Horseshoe crab hemocyte polypeptides, and

preparation and DNA encoding thereof

Iwanaga, S., Kawabata, S..

Saito, T. 1999/01/19 Horseshoe crab [71]

US5166321 Cecropin polypeptides with activity against

Gram-positive and Gram-negative bacteria

Lai, J.S., Lee, J.H.,

Callaway, J.E. 1999/24/11

Modified

cecropin of

H.cecropia

[72]

EP1146052 Arthropod defensins Presnail, J.K., Weng, Z.,

Wong, J.F.H. 1982/08/19

Scalopendra

canidens DS [73]

US6911524 Antimicrobial peptides derived from mollusks Roch, P., Mitta, G., Hubert, F.,

Noel, T. 2005/05/28

Bivalve

mollusc

shellfish

[74]

WO035677 Antimicrobial peptides from the venom of the

spider Cupiennius salei

Schaller, J., Walz, A., Nentwig,

W., Kuhn-Nentwig, L. 2003/05/01

Cupiennius

salei (Spider) [75]

which are found in every insect species investigated to date (Table 4, EP1146052). Defensins are larger than cecropins with a size of 33 – 46 residues; and three to four internal disulfide bridges stabilize their structure. [2] The activities of the invertebrate defensins can be divided according to whether their main biological activity is directed toward bacteria or fungi [7].

VERTEBRATES

Antimicrobial peptides have been isolated from a wide range of vertebrate species, including fish, amphibians, and mammals, indicating that, even in the presence of an adaptive immune response, these peptides have an important role in host defense [7].

8 Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 Rizza et al.

The activities of these peptides likely contribute to the first line of defense, especially where they are found in very high concentrations, such as in the granules of phagocytic cells or the crypts of the small intestine. [73] Beyond their antimicrobial function, the antimicrobial peptides are known to be multi-functional (see Table 5 [74-92]). These peptides are important effectors molecules of the innate immune system and inflammatory responses. They are able to enhance phagocytosis, stimulate prostaglandin release, neutralize the septic effects of LPS shed from Gram-negative bacteria, promote recruitment and accumulation of various immune cells at inflammatory sites, promote angiogenesis, and induce wound repair. [7] In fact, it has been observed that a single antimicrobial peptide can exhibit all of these functions [93,94].

Consistent with their role in direct and indirect antimic-robial defenses, antimicrobial peptides in vertebrates are found at sites that routinely encounter pathogens, such as mucosal surfaces and the skin, as well as within granules of immune cells. [7] In mammals, including humans, AMPs are produced in both circulating defensive cells and on epithelial surfaces [95]. By rapidly killing a broad spectrum of microbes, AMPs play a ‘frontline’ role in the defense of epithelial barriers. Many thick, multilayered epithelial surfaces, such as epidermis and the oral mucosa, tolerate the attachment of microbes on the dead superficial layers of epithelium (which are continuously being sloughed), bacteria, and cells together. [12]

The -helical magainins are the prototypic amphibian antimicrobial peptides, with strong membrane-permea-bilizing activity towards Gram-positive and Gram-negative bacteria, fungi, yeasts, and viruses. In addition to their presence in the skin, amphibian antimicrobial peptides are produced in the mucosa of the stomach, indicating a role in protection from ingested pathogens. [96] The magainins, originally isolated from the skin of the African frog Xenopus laevis [9,97] were the first molecules used to evaluate their biomedical applications (http://www.genaera.com; http:// www.inimexpharma.com). For example, the magainins and their analogs have been found capable of lysing both hematopoietic tumor and solid tumor cells with little toxic effect on normal blood lymphocytes [98]. Temporin L, an AMP isolated from the skin of the frog Rana temporaria, induce necrosis in tumor cells [99].

In general, the AMPs of vertebrates are synthesized as large precursors, containing one or multiple copies of the active peptide segment which are released by proteolytic processing. In the simplest cases the cotranslational removal of an N-terminal signal peptide releases the active moiety but more commonly one or more anionic propieces are also removed during processing. [100,101]

For example, the cathelicidins, a large and diverse group of vertebrate antimicrobial peptides, is frequently proteoly-tically cleaved from the highly variable C-terminal antimic-robial domain [102,103]. Most cathelicidins are stored in an inactive propeptide state, mostly within granules of circulating immune cells. Neutrophil secretory granules are the predominant source of cathelicidins, but they may also be expressed in mucosal surfaces in the mouth, lungs, and genitourinary tract and in skin keratinocytes in inflammatory

disorders. [104] The cathelicidin family components are characterized by a diverse C-terminal antimicrobial domain connected to a conserved cathelin-like N-terminal domain – the propiece – of approximately 100 residues, that is proteo-lytically cleaved to generate the mature, active peptide contained within the C terminus [102,103]. Beyond the common N terminus, the cathelicidins are heterogeneous by size, ranging from 12 to 80 amino acid residues, and composition, and fit several distinct structural classes, with

-helical, -hairpin, and proline/arginine-rich peptides all represented. [103] The structural diversity within the cathelicidin family is also indicative of their apparently distinct functions, and they exhibit a diverse spectrum of microbicidal and immunomodulatory activities [104]. Cathelicidins have been subject to patents in different mechanisms of action, e.g. inducing its expression (see Table

5, WO136159; WO076162 [105] and WO040192 [106]), as fusion products of biocides (Table 5, US147442).

A second prominent and highly complex group of mammalian antimicrobial peptides is the defensins, a group of cyclic cysteine-rich peptides [107]. As with cathelicidins, vertebrate defensins are synthesized as prepeptides which require proteolytic processing to their active peptide forms [7]. They are categorized into three subfamilies [23] on the basis of their size and pattern of disulfide bonding: - (e.g. WO008162) [108], - (e.g. WO092309, see Table 5) and -defensins (e.g. WO105883 [109] and US0022829 [110]). - and -defensins are found in neutrophils, epithelial surfaces, certain macrophage populations and Paneth cells (specialized granule-rich intestinal host defense cells) [22,95], where they act as immunomodulators. For example, the -defensins are almost certainly bactericidal at the high (mgmL

-1) concen-

trations found in neutrophils granules, but they probably act primarily as immunomodulators at lower concentrations released by degranulation at inflammatory sites [7,95]. The

-defensins can also bind to chemokine receptors, stimula-ting dendritic cells and T cells; so there are also immuno-modulatory peptides [10]. The Defensins Knowledgebase (http://defensins.bii.a-star.edu.sg/) is related to defensins in a broad sense consideration including material products (defensins or defensin-like peptides or nucleic acid sequences, synthetic or natural) or for novel methods of detecting, quantization, synthesizing or delivering AMPs and nucleic acid in general. Nineteen of these patents have been published in 2006 and twenty seven have been published in 2007. Several patents refer to animal (Table 5, WO142542, WO002520 and US147442) and human applications (Table

5, WO081486, WO047512, WO084131 and US147442).

MODE OF ACTION

The antimicrobial activity and specificity of the AMPs depends on a number of parameters such as the amino sequence, the peptide size, membrane lipids and peptide concentration [23,111]. They display a broad spectrum of activity, that not only kill bacteria, but are also cytotoxic to fungi [16-18,112], protozoa [20], malignant cells [98], and even enveloped viruses like vesicular stomatitis virus (VSV), influenza virus, herpes simplex virus and human immunodeficiency virus (HIV-1) [5,21,94].

Fundamental differences exist between microbial and host membranes that represent potentially selective targets

Biomolecules as Host Defense Weapons Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 9

Table 5. Selection of Relevant Patents Covering AMPs from Vertebrates

Publication

number Title Inventors

Publication

date Source

Ref.

No.

US6329340 Human defensin DEF-X Bougueleret, L.,

Chumakov, I. 2001/11/12 Homo sapiens [81]

WO142542 Cationic milk proteins for treating mastitis Bragger, J.M. 2007/12/13 Bovines [82]

WO084131

Furin inhibitors and alpha-defensins for the

treatment or prevention of papillomavirus

infection

Day, P.M., Richards, R.,

Buck, C., Schiller, J.T.,

Lowy, D.R.

200/08/10 Homo sapiens [83]

WO047512 Inhibition of PAX2 by DEFB-1 induction as a

therapy for cancer Donald, C.D. 2007/04/26 Homo sapiens [84]

US7348409 Antimicrobially active peptide Garbe, C., Schittek, B. 2008/02/25 Homo sapiens [85]

US147442 Biocides

Homan, J., Imboden, M.,

Riggs, M., Carryn, S.

Schaefer, D.A.

2006/06/07 Synthetic [86]

US7186795 Lactoferricin gene and transformant expressing

lactoferricin Kim, H., Kang, D., Lee, J. 2007/06/03 Bovines [87]

WO136159 Sphingosylphosphorylcoline antagonist for

restoration of antimicrobial peptides

Kim, J.H., Kim, H., Sung,

K.S., Kim, D.K., Sung, D.S.,

Park, J.H., Cho, S.A., Kim,

K.M., Lee, C.H., Kim, J.J.

2007/11/29 Homo sapiens [88]

US5635594 Gallinacins - antibiotic peptides Lehrer, R.I., Kokryakov,

V.N., Harwig, S.S. 1997/03/06 Gallus gallus [89]

US7314858 Retrocyclins: antiviral and antimicrobial peptides Lehrer, R.I., Waring, A.J.,

Cole, A.M., Hong, T.B. 2008/01/01

Orangutan,

Gorilla,

Homo sapiens

[90]

WO092309 Human Beta-defensin (HBD-3), a highly cationic

beta-defensin-3 antimicrobial peptide

McCray Jr., P.B., Tack, B.F.

Jia, H.P., Schutte, B.C. 2001/12/06 Homo sapiens [91]

US6753403 Antimicrobial peptides isolated from fish Noga, E.J., Silphaduang, U. 2004/06/22 Fish [92]

US7223840 Antimicrobial peptide Olsen, H.S., Ruben, S.M. 2007/05/29 Homo sapiens [93]

US6211148 Antimicrobial peptides from bovine neutrophils Selsted, M.E., Cullor, J.S. 2001/04/03 Bovines [94]

US6696559 Antimicrobial peptides and methods of use Selsted, M.E. 2004/02/24 Bovines [95]

WO002520

The use of porcine beta-defensin-1 for treating or

peventing a microbial infection in a vertebrate

subject

Shokrollah, E., Gerdts, V.,

Babiuk, L. 2006/01/12 Porcines [96]

WO081486 Oral administration of defensins to treat intestinal

diseases

Wehkamp, J., Huang, N.,

Bevins, C.L., Stange, E. 2007/07/19 Homo sapiens [97]

WO007873 Production and use of tracheal antimicrobial

peptides

Zasloff, M, Bevins CL,

Diamond G. 1992/05/14 Bovines [98]

WO018516 Novel endopeptidase Zasloff, M., Resnick, N.M. 1992/10/29 Xenopus laevis [99]

10 Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 Rizza et al.

for antimicrobial peptides, best understood for bacterial targets. Bacterial membranes are organized in such a way that the outermost leaflet of the bilayer, the surface exposed to the outer world, is rich in anionic phospholipids [22]. Conversely, the cell membranes of plants and animals are rich in phospholipids with no net charge; most of the lipids with negatively charged headgroups are segregated into the inner leaflet, facing the cytoplasm. In the case of animal’s membranes, the presence of cholesterol in general reduces the activity of antimicrobial peptides, due either to stabilization of the lipid bilayer or to interactions between cholesterol and the peptide [8,22]. These differences explains why the concentration necessary to kill eukaryotic cells are much higher than those required for killing most bacteria [22].

A variety of techniques - microscopy, studies with model membranes [113], circular dichroism [114], nuclear magne-tic resonance (NMR) spectroscopy [115-120], neutron diffraction [121], etc. - have been used to assess the mecha-nisms of antimicrobial peptide activity. However, no single technique is capable of adequately determine the mechanism of action of the peptides [111]. These techniques indicated that the AMPs interact with membranes [23,24,122]. The last studies divided these peptides into two mechanistic classes: 1) membrane disruptive (barrel-stave, toroidal pore and carpet models), and 2) non-membrane disruptive peptides (intracellular targets) [23].

Regardless of the time required, or the specific antimicrobial mechanism, specific steps must be followed in order to induce pathogen killing [123].

1) Attraction between the peptide and the target cell, pathogen surfaces [124], is meant to occur through electrostatic bonding between the cationic peptide and negatively charged components present on the outer bacterial envelope, such as phosphate groups within the LPS of Gram-negative bacteria or lipoteichoic acids present on the surfaces of Gram-positive bacteria [125,126].

2) Binding of the peptide to the membrane [127]. In the case of Gram-negative bacteria, the cationic peptides interact with the highly negatively charged outer membrane by hydrophobic interactions. The peptides initially interact with the polyanionic surface LPS and competitively displace the divalent cations (magnesium and calcium) that bridge and partly neutralize the LPS [6]. Addition of cationic peptides results in displacement of metal ions, facilitating the formation of destabilized areas through which the peptide translocates the outer membrane in a process termed self-promoted uptake. [128]

3) Peptide insertion and membrane permeability [129,130]. At high AMPs concentrations, the peptides are orientated perpendicularly and inserted into the bilayer, forming transmembrane pores, this lead to a lethal increase in the permeability of the cell membrane [25].

At date, several prominent models have been proposed to explain membrane permeabilization, even if it should be stated that there is not universal consensus among investi-gators in this matter. Each one of these indicate a different

type of intermediate that can lead to one of the three types of events: formation of a transient channel, micellarization or dissolution of the membrane, or translocation across the membrane. As a result, the peptide can permeabilize the membrane and/or translocate across the membrane and into the cytoplasm without causing any major membrane disrup-tion. [130] This supports the idea that membrane permeabi-lization, while required, may not be sufficient to cause cell death [36].

THE BARREL-STAVE MODEL

Ever since its introduction in the early 1970s, the ‘barrel-stave model’ has been viewed as the prototype of peptide-induced transmembrane pores [131]. One example as a barrel-stave model is the pore induced by alamethicin, which is a peptide antibiotic produced by Trichoderma viride [132]. In this model, there is a perpendicular insertion of a variable number of peptides in the membrane. The peptides form a bundle inside the membrane leading to a transmembrane pore or channel with a cylindrical structure, also referred as staves in a barrel-like ring. [131] The ‘stave’ term refers to individual transmembrane spokes within this barrel, which may be composed either by of individual peptides or peptide complexes [127]. In this mechanism, the hydrophobic surfaces of -helical or -sheet peptides align with the core region of the bilayer, whereas the hydrophilic peptide regions form an aqueous pore lining [132,133]. A minimal length of 22 amino acids is required to transverse the lipid bilayers with -helical peptides, or 8 amino acids in case the peptide adopts a -sheeted structure, while lengths are comparable to the thickness of the hydrocarbon of a phospholipid bilayer ( 3 nm) [134].

THE TOROIDAL PORE MODEL O WORMHOLE MECHANISM

The toroidal pore model is one of the most well-characterized models [68,77,81]. Differing from the barrel-stave model since the peptides are intercalated with lipid head groups in the transmembrane channel [131]; while this structure has been referred to as a supramolecular complex [127]. In this model, peptides in the extracellular environ-ment take on a -helical structure as they insert into the membrane. The hydrophobic residues of the peptides displace the polar head groups, creating a breach in the hydrophobic region and inducing a positive curvature strain in the membrane. [135,136] The model postulates that peptide and lipid together, form well-defined pores, so that the water core is lined both the inserted peptides and the lipids head group [137,138]. The introduction of strain makes the membrane more vulnerable to ensuing peptide interactions [127].

AMPs like the magainins [137-140], protegrins [115], melittin [141] and LL-37 [131,139,142] are proposed to use this mechanism as their mode of action.

THE CARPET MECHANISM

This mechanism explains the activity of some antimic-robial peptides, which act against microorganisms through a relative diffuse manner, likes membrane deter-gents. In this model, a high density of peptides accumulates on the target bilayer surface [143]. The AMPs ovispirin [116] and

Biomolecules as Host Defense Weapons Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 11

cecropin P1 [144], that orientate parallel to the membrane target, explains this model. Peptides initially bind to the anionic phospholipid head groups by electrostatic inter-actions, covering the surface of the membrane in a carpet-like manner [111]. At high peptide concentrations, surface-oriented peptides are meant to disrupt the bilayer in a detergent-like manner, eventually leading to the formation of micelles [145]. Phospholipid displacement changes in membrane fluidity and/or reductions in membrane barrier properties subsequently lead to membrane disruption. A specific quaternary structure in this mechanism is not neces-sary. Thus, when a threshold peptide density or concen-tration is reached, the membrane is subjected to an unfa-vorable energetic, and membrane integrity is lost [128]. There is no channel formation, and peptides do not necessarily insert themselves into the hydrophobic membrane core [127].

OTHER MECHANISMS OF ACTION: INTRA-CELLULAR TARGETS

Initially, AMPs were meant to act only at the plasma membrane although some exert their cytotoxic effects via interaction with intracellular targets. Peptides that do not seem to act on membranes are meant to act on cytoplasmic targets, supporting the idea of additional or complementary mechanisms. Translocation across the cytoplasmic memb-ranes, without causing significant leakage of intracellular contents or loss of transmembrane potential, is intended to occur by a process of micellar aggregate mechanism. [5,36,146]

In this case, the mechanism of action of AMPs acts interfering with intracellular processes: inhibition of DNA synthesis in Escherichia coli (indolicidin, [147]), inhibition of the activity of the cytoplasmic prokaryotic heat shock chaperone DnaK (proline-rich insect AMPs, [148,149]), inducing reactive oxygen species in fungi (NaD1, [36]) etc.

These data support the hypothesis that AMPs can penetrate the pathogen cells despite the interaction with the cytoplasmic membrane.

IS THERE AN INDUCED RESISTANCE TO AMPS?

Microbial pathogens occupy and exploit a diverse variety of tissues and niches where they must confront with host defenses to survive [127]. Considering that AMPs are natural barriers to pathogens infections, certain microbial pathogens ought to have developed a variety of strategies which render them more resistant to AMPs due to stable structural or functional properties [23].

Direct degradation of antimicrobial peptides is a strategy used by the Gram-negative bacteria to resist the bactericidal activity of antimicrobial peptides. This strategy is dependent on the production of outer membrane-associated proteases [150,151], which cleave antimicrobial peptides outside the cells and enable bacteria to evade killing. For instance, it has been found that E. coli outer membrane protease OmpT hydrolyzes the antimicrobial peptide protamine before penetrating this bacterium [152]. Other resistant species, such as Porphyromonas gingivalis, secrete digestive proteases that destroy peptides [8].

Other pathogens have evolved other countermeasures to limit AMPs effectiveness, such as chemical modifications of cell surface properties and/or alteration in outer membrane components [111]. For example, some strains of Klebsiella pneumoniae are more resistant to some AMPs; the mecha-nism of resistance is dependent on the bacterial capsule polysaccharide (CPS), which protects bacteria by limiting the interaction of AMPs with the membrane targets [153].

Antimicrobial resistance is also associated with the alteration of energy-dependent pumps at the membrane level [154].

APPLICATIONS OF AMPS

IN THE CLINIC

In several cases, the antimicrobial peptides show an increased action against antibiotic resistant bacteria. A high number of patents document this (e.g. US0003938 [155], US6906035 [156], US6191254 [157], WO080625 [158], WO000694 [159] and WO054314 [160]).

In addition to the benefits of improved effects of antibacterial therapy, evaluation of the synergic actions of antimicrobial agents may be used to explore possible modes of action of new antibiotics [161]. As discussed above, the mechanism of action of peptides leads to the disruption of the outer membrane barrier and/or perturbation of the cytoplasmic membrane or via interaction with intracellular targets. Possibly as a consequence of these actions, antimic-robial peptides can demonstrate synergy with conventional antibiotics against both Gram-negative [96] and Gram-positive [162] bacteria, including multi-drug resistant antibiotic. Some of these approaches have been patented (e.g. WO045156 [163], WO050611 [164], US5981844 [165]).

A second type of synergy that has been observed is between naturally peptides [129]. This was first demons-trated [166] with the antimicrobial peptides magainin 2 and PGLa, two naturally occurring peptides from the same host (see US5254537 [167]). Other experiments, demonstrated that mammalian peptides from different structural classes frequently show synergy with each other and selectively show synergy with human lysozyme in a peptide-specific manner [168].

Because of the increasing antibiotic resistance problem, there is a need to develop new classes of antibiotics. In order to be a good candidate for therapeutic use, a drug needs to show good activity, appropriate function, low toxicity, posses stability in vivo and also be reasonably inexpensive to manufacture [4]. Cationic antimicrobial peptides contain many of the desirable features of a nobel antibiotic class [6]. In particular they have a broad spectrum of activity, kill pathogen rapidly, are unaffected by classical antibiotic resistance mutations, do not easily select antibiotic resistant variants, show synergy with classical antibiotics, neutralize endotoxin, and are active in animal models. [6] Despite this, many problems that limit the systemic use of these compounds must be solved. For instance, some AMPs are very toxic for mammalian cells, whereas others show little or no acute cytotoxicity [6,26]. Currently research is being done to structurally alter these compounds to make them less toxic

12 Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 Rizza et al.

to the consumer [4]. Cost is another issue, since these peptides have relatively high molecular weights compared with most antibiotics and will have to be produced recombinantly to keep prices down [26]. Another problem would be their lability to proteases in the body. In this regard, there are strategies for protecting the peptides from proteases, including liposomal incorporation or chemical modification [6] (see patent WO079185 [169]).

IN BIOTECHNOLOGY

Antimicrobial peptides have a number of applications in biotechnology. In the Defensins Knowledgebase (http:// defensins.bii.a-star.edu.sg/) besides patents covering several modes of applications e.g. topical use (see Table 5, WO136159; WO144613 [170] and WO081487 [171]), vaccines (Table 5, WO002520; and WO133373 [172]), oral formulations (WO038623 [173]) there are others revealing efforts leading to enhance their uses e.g. surface-layer protein-coated microspheres for the delivery of a therapeutic agent to the intestine (WO087557 [174]), production of human alpha-defensins in plant cells (WO081487 [171]), prevention of growth of microorganisms on medical devices (US003538 [175], WO094579 [176] and WO058752 [177]).

Other of the applications for cationic peptides would be the formation of transgenic plants. Phytopathogens are a major problem in cultivated and stored crops leading to the loss of between 30-50 billion dollars annually [4]. Pesticides and antimicrobials are not really a good way to combat phytopathogens because they can be expensive, detrimental to the environment and harmful to the consumer. They can also lead to the development of resistant bacterial strains. It is proved that by producing transgenic plants that express cationic peptides with broad specificity, many of these problems will be circumvented [15,51,178]. Some approa-ches to this are described in patents referred WO006079 [179], WO062927 [180] and WO006564 [181], which focuses in the achievement of resistance to phytopathogens including both fungi and bacteria; bacteria as diverse as Erwinia, Pseudomonas, and Xanthomonas, and fungi as diverse as Botrytis and Phytophthora.

In recent years, packaging research has focused more on biodegradable films, including films made from plant and animal edible protein sources such as corn, wheat gluten, soy and peanut protein, cotton seed, albumin, gelatin collagen, casein and whey proteins. Several compounds have been proposed for antimicrobial activity in food packaging, including organic acids, enzymes such as lysozyme, and also fungicides. Synthetic antimicrobial peptides and natural compounds, such as polycyclic peptides like nisin and spices rich in phenolic compounds, have been studied as potential food preservatives added to the edible films which are safe for human consumption. [182,183]

Animal feeds are sometimes used as a means to introduce antibiotics and other growth-promoting compounds. Recent concerns about antibiotic resistance have spurred a search for alternatives. AMPs expressed in seed coats could provide a viable substitute. They are non-immunogenic when ingested but may stimulate an immune response if injected. Since seed coats have reduced digestibility when compared to meal, the active AMP inside the hourglass cell may be

protected from proteolytic activity while the seed coat slowly breaks down, releasing AMP in a similar manner to a slow-release pill [184].

CURRENT & FUTURE DEVELOPMENTS

The discovery of the widespread distribution of anti-microbial peptides over the past 20 years has provided insights into the innate defensive systems that permit multicellular organisms, including humans, in order to live in harmony with microbes. It is hard to imagine that most ani-mals now alive, including insects and creatures like octopus and starfish, rely heavily on antimicrobial peptides for their defense against microbes, and do so quite effectively without the help of lymphocytes, a thymus, or antibodies. [8]

In plants, over 300 putative defensive-like genes have been identified in Arabidopsis and Medicago [36] and many gene constructions including sequences coding for AMPs have been expressed on model or crop plants [3].

Newly characterized molecules have inspired molecular designs for the creation of therapeutics, and will continue to do so as more are being discovered, because of being based on antimicrobial strategies that have proven efficacious over millennia. Studies both in the laboratory and in the clinic confirm that emergence of resistance against antimicrobial peptides is less likely than observed for conventional antibio-tics, and provides the impetus to develop antimicrobial pep-tides, both natural and laboratory conceived, into therapeu-tically useful agents. [8] Another plus of antimicrobial peptides, is that they vary between organisms, which could lead to a greater selection of drugs. It may also be possible to transgenically alter plants and animals in agriculture so that they may posses increased levels of AMPs in an attempt of reducing losses Developments of biomolecules suitable for therapeutic scopes and agricultural use in plant protection have several challenges such as intrinsic toxicity, in vivo stability and suitable formulation in harmony to cost of production. In this sense, biotechnological procedures using microbial systems or transgenic crop as plant factories might help to solve these challenges.

Thus, designing antimicrobial peptides able to induce transmembrane pores and/or intracellular targets and with the capacity to have synergistic effects with other host innate immune molecules, will facilitate the development of more efficient weapons against microbial pathogens.

ACKNOWLEDGEMENTS

P.D.D. was the recipient of a Doctoral Studentship from Agencia Nacional de Investigación e Innovación (ANII - Uruguay). The authors wish to acknowledge María Cristina Martin for her help in preparing this review. This work was supported by INIA, Uruguay, and PEDECIBA Química, Uruguay.

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