Biomolecules as Host Defense Weapons Against ...

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

1

Biomolecules as Host Defense Weapons Against Microbial Pathogens Marco Dalla Rizza1,*, Paola Díaz Dellavalle1, Rafael Narancio1, Andrea Cabrera1 and Fernando 2 Ferreira 1

Biotechnology 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: [email protected]

1872-2156/08 $100.00+.00

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 molecules, 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 pathogenassociated 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 protection products. [3] The knowledge acquired in the past two decades and the discovery of new groups of antimicrobial peptides, make © 2008 Bentham Science Publishers Ltd.

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

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 powerful arsenal of molecules that could be the antimicrobial drugs of the new century as an innovative response to the increasing 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 Gramnegative 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

Rizza et al.

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 CATIONIC PEPTIDES

OF

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 peptides have a broad spectrum of antimicrobial activity including activity against both Gram-negative and Grampositive 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 amphipathic 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 glycosylation, carboxi-terminal amidation, amino-acid isomerization, 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

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 characterized 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,

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

3

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 invertebrates 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 hemocytes), 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 membrane-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 suppressing HIV-1 gene expression [71]. Some C-terminus modifications (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,


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]



(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]

Bacteria strain and bacteriocin

Fliss, I., Desbiens, M., Lacroix, L., Tahiri, I., Benech, R., Kheadr, E.

2007/07/24

Carnobacterium divergens

[37]

US7247306

produced therefrom

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]

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]

US6989370

5


Table 3.

Rizza et al.

Selection of Relevant Patents Covering AMPs from Plants

Publication number

Title

US6150588

DNA encoding antimicrobial proteins from Impatiens

US5750504

Ref.

Publication date

Source

Attenborough, S., Broekaert, W.F., Osborn, R.W., Ray, J.A., Rees, S.B., Tailor, R.H.

2000/11/21

Impatiens balsamina

[44]

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

1995/05/18

Zea mays

[47]

2007/07/03

Dimorphotheca, Picramnia, Parthenium, Nicotiana, Vernonia, and Helianthus

[48]

2005/06/21

Macadamia integrifolia

[49]

2006/06/27

Macadamia integrifolia

[50]

Inventors

Duvick, J.P., Rood, T.A., Rao, A.G. Famodu, O.O., Herrmann, R.,

US7238781

Plant defensin polynucleotides and methods thereof

Lu, A.L., McCutchen, B.F., Miao, B.F., Miao, G., Presnail, J.K., Weng, Z.

US6909032

DNA encoding a Macadamia integrifolia anti-microbial protein, constructs comprising the same and plant material comprising the constructs

US7067624

Antimicrobial proteins

No.

Manners, J.M., Marcus, J.P., Clifford, K., Green, J.L., Harrison, S.J.

Manners, J.M., Marcus, J.P., Goulter, K.C., Green, J.L., Bower, N.I.

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

2006/08/03

Cicer arietimun

[53]

2005/03/08

Raphunus sativus

[54]

Zea mays, Oryza sativa, Triticum aeativum, Glycine max, Beta vulgaria, Hedera helix, Tulipa fosteriana, Tulipa gesneriana, Cyamopsis tetragonoloba

[55]

Reddy, V.S., Hassari, A., Isalm, A.

US6864068

Antifungal proteins

Rees, S.B., De Samblanx, G.W., Broekaert, W.F.

Simmons, C.R., Navarro Acevedo, P.A., Harvell, L., US6911577

Defensin polynucleotides and methods of use

Cahoon, R., McCutchen, B.F., Lu, A.L., Herrmann, R., Wong, J.F.H.

2005/06/28



7

(Table 3) Contd….

Publication number

EP1849868

Title

Inventors

Weng, Z., Miao, G.,

Plant defensins

Publication date

Source

2008/02/20

Dimorphotheca, Picramnia, Parthenium, and Nicotiana

Famodu, O.O.

US6086885

Table 4.

Anti-bacterial protein extracts from seeds of marigold and paprika

Ziegenfus, S., Brinkhaus, F., Greaves, J.

2001/01/17

Ref. No.

Tagetes,

[56]

[57]

Capsicum

Selection of Relevant Recent Patents Covering AMPs from Invertebrates Ref.

Publication number

Title

Inventors

Publication date

Source

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

US4355104

Bacteriolytic proteins

Hultmark, D., Steiner, H.,

Horseshoe crab hemocyte polypeptides, and preparation and DNA encoding thereof

Iwanaga, S., Kawabata, S..

US5166321

Cecropin polypeptides with activity against Gram-positive and Gram-negative bacteria

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

EP1146052

Arthropod defensins

[69]

(Crustacean)

1982/08/19

Hyalophora cecropia (Insect)

[70]

1999/01/19

Horseshoe crab

[71]

1999/24/11

Modified cecropin of H.cecropia

[72]

1982/08/19

Scalopendra canidens DS

[73]

Rasmuson, U., Boman, H.G.

US5861378

Penaeid prawns

No.

Saito, T.

Callaway, J.E. Presnail, J.K., Weng, Z., Wong, J.F.H.

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].


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 antimicrobial 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-permeabilizing 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 proteolytically cleaved from the highly variable C-terminal antimicrobial 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

Rizza et al.

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 proteolytically 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) concentrations 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, stimulating dendritic cells and T cells; so there are also immunomodulatory 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


Table 5.


Selection of Relevant Patents Covering AMPs from Vertebrates

Publication number

Title

US6329340

Human defensin DEF-X

Inventors Bougueleret, L.,

Ref.

Publication date

Source

2001/11/12

Homo sapiens

[81]

2007/12/13

Bovines

[82]

200/08/10

Homo sapiens

[83]

No.

Chumakov, I. WO142542

Cationic milk proteins for treating mastitis

Bragger, J.M. Day, P.M., Richards, R.,

WO084131

Furin inhibitors and alpha-defensins for the treatment or prevention of papillomavirus infection

Buck, C., Schiller, J.T., Lowy, D.R.

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

2006/06/07

Synthetic

[86]

Homan, J., Imboden, M., Riggs, M., Carryn, S. Schaefer, D.A. 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

2008/01/01

Orangutan, Gorilla,

[90]

Lehrer, R.I., Waring, A.J., Cole, A.M., Hong, T.B.

Human Beta-defensin (HBD-3), a highly cationic beta-defensin-3 antimicrobial peptide

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

US6753403

Antimicrobial peptides isolated from fish

US7223840

WO092309

Homo sapiens 2001/12/06

Homo sapiens

[91]

Noga, E.J., Silphaduang, U.

2004/06/22

Fish

[92]

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.,

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]

Jia, H.P., Schutte, B.C.

Babiuk, L.

9


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 magnetic resonance (NMR) spectroscopy [115-120], neutron diffraction [121], etc. - have been used to assess the mechanisms 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 investigators in this matter. Each one of these indicate a different

Rizza et al.

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 disruption. [130] This supports the idea that membrane permeabilization, while required, may not be sufficient to cause cell death [36]. THE BARREL-STAVE MODEL Ever since its introduction in the early 1970s, the ‘barrelstave model’ has been viewed as the prototype of peptideinduced 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 wellcharacterized models [68,77,81]. Differing from the barrelstave 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 environment 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 antimicrobial 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



cecropin P1 [144], that orientate parallel to the membrane target, explains this model. Peptides initially bind to the anionic phospholipid head groups by electrostatic interactions, covering the surface of the membrane in a carpetlike manner [111]. At high peptide concentrations, surfaceoriented 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 necessary. Thus, when a threshold peptide density or concentration is reached, the membrane is subjected to an unfavorable 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 CELLULAR TARGETS

OF

ACTION:

INTRA-

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 membranes, 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].

11

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 mechanism 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, antimicrobial peptides can demonstrate synergy with conventional antibiotics against both Gram-negative [96] and Grampositive [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 demonstrated [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


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 approaches 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

Rizza et al.

protected from proteolytic activity while the seed coat slowly breaks down, releasing AMP in a similar manner to a slowrelease pill [184]. CURRENT & FUTURE DEVELOPMENTS The discovery of the widespread distribution of antimicrobial 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 animals 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 antibiotics, and provides the impetus to develop antimicrobial peptides, both natural and laboratory conceived, into therapeutically 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. REFERENCES [1]

[2]

Marshall SH, Arenas G. Antimicrobial peptides: A natural alternative to chemical antibiotics and a potential for applied biotechnology. Electronic J Biotech [serial online] 2003 Aug 15 [cited 2007 September 11]; 6: [about 14 pages]. Available from: http://www.bioline.org.br/pdf?ej03030 Bulet P, Stöcklin R. Insect antimicrobial peptides: structures, properties and gene regulation. Protein Pept Lett. 2005; 12: 3-11.

Biomolecules as Host Defense Weapons [3] [4] [5] [6] [7] [8] [9]

[10]

[11]

[12] [13] [14] [15]

[16] [17]

[18] [19] [20]

[21] [22] [23]

[24] [25] [26] [27] [28]

[29]

[30]

Montesinos E. Antimicrobial peptides and plant disease control. FEMS Microbiol Lett. 2007; 270: 1-11. Wilcox S. The new antimicrobials: cationic peptides. BioTeach Journal. 2004; 2: 88-91. Powers JPS, Hancock REW. The relationship between peptide structure and antibacterial activity. Peptides. 2003; 24: 1681-1691. Hancock REW, Scott MG. The role of antimicrobial peptides in animal defenses. PNAS. 2000; 97: 8856-8861. Jenssen H, Hamill P, Hancock REW. Peptide antimicrobial agents. Clin Microbiol Rev. 2006; 19: 491-511. Zasloff M. Antimicrobial peptides of multicellular organisms. Nature. 2002; 415: 389-395. Zasloff M. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc Natl Acad Sci USA. 1987; 84: 5449-5453. Chan DI, Prenner EJ, Vogel HJ. Tryptophan- and arginine-rich antimicrobial peptides: structures and mechanisms of action. Biochim Biophys Acta. 2006; 1758: 1184-1202. Giangaspero A, Sandri L, Tossi A. Amphipathic helical antimicrobial peptides. A systematic study of the effects of structural and physical properties on biological activity. Eur J Biochem. 2001; 268: 5589-5600. Zasloff M. Inducing endogenous antimicrobial peptides to battle infections. Proc Natl Acad Sci USA 2006; 13: 8913-8914. De Lucca AJ, Cleveland TE, Wedge DE. Plant-derived antifungal proteins and peptides. Can J Microbiol. 2005; 51: 1001-1014. De Lucca AJ, Walsh TJ. Antifungal peptides: novel therapeutic compounds against emerging pathogens. Antimicrob Agents Chemother. 1999; 43: 1-11. Huynh QK, Hironakan CM, Levinell EB, et al. Antifungal proteins from plants: purification, molecular cloning, and antifungal properties of chitinases from maize seed. J Biol Chem. 1992; 267: 6635-6640. Selitrennikoff CP. Antifungal proteins. Appl Environ Microbiol. 2001; 67: 2883-2894. Terras FRG, Schoofs HME, De Bolle MFC, et al. Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem. 1992; 267: 15301-15309. Terras FRG, Torrekens S, Van Leuven F, et al. A new family of basic cysteine-rich plant antifungal proteins from Brassicaceae species FEBS Lett. 1993; 316: 233-240. Vigers AJ, Roberts WK, Selitrennikoff CP. A new family of plant antifungal proteins. Mol Plant Microbe Interact. 1991; 4: 315-323. Aley SB, Zimmerman M, Hetsko M, Selsted ME, Gillin FD. Killing of Giardia lamblia by cryptdins and cationic neutrophil peptides. Infect Immun. 1994; 62: 5397-5403. Kliger Y, Gallo SA, Peisajovich SG, et al. Mode of action of an antiviral peptide from HIV-1: inhibition at a post lipid-mixing stage. J Biol Chem. 2001; 276: 1391-1397. Ganz T. The role of antimicrobial peptides in innate immunity. Integr Comp Biol. 2003; 43: 300-304. Giuliani A, Pirri G, Fabiole Nicoletto S. Antimicrobial peptides: an overview of a promising class of therapeutics. C Europ J Biol. 2007; 2: 1-33. Hancock REW, Chapple DS. Peptide antibiotics. Antimicrob Agents Chemother. 1999; 43: 1317-1323. Van´t Hof W, Veerman EC, Helmerhorst EJ, Amerongen AV. Antimicrobial peptides: properties and applicability. Biol Chem. 2001; 382: 597-619. Hancock REW, Lehrer RI. Cationic peptides: a new source of antibiotics. Trends Biotechnol. 1998; 16: 82-88. Hewish DR, Barnham KJ, Werkmeister JA, et al. Structure and activity of D-Pro14 melittin. J Protein Chem. 2002; 21: 243-253. Gesell J, Zasloff M, Opella SJ. Two-dimensional 1H NMR experiments show that the 23-residue magainin antibiotic peptide is an -helix in dodecylphosphocholine micelles, sodium dodecylsulfate micelles, and trifluoroethanol/water solution. J Biomol NMR. 1997; 9: 127-135. Hwang PM, Zhou N, Shan X, Arrowsmith CH, Vogel HJ. Threedimensional solution structure of lactoferricin B, an antimicrobial peptide derived from bovine lactoferrin. Biochemistry. 1998; 37: 4288-4298. Bauer F, Schweimer K, Kluver E, et al. Structure determination of human and murine beta-defensins reveals structural conservation in


[31]

[32]

[33]

[34] [35] [36] [37] [38] [39] [40]

[41] [42] [43]

[44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55]

[56] [57] [58]

[59]

[60] [61] [62]

[63]

13

the absence of significant sequence similarity. Protein Sci. 2001; 10: 2470-2479. Fant F, Vranken W, Broekaert WF, Borremans FA. Determination of the three-dimensional solution structure of Raphanus sativus antifungal protein 1 by 1H NMR. J Mol Biol. 1998; 279: 257-270. Mandard N, Sodano P, Labbe H, et al. Solution structure of thanatin, a potent bactericidal and fungicidal insect peptide, determined from proton two-dimensional nuclear magnetic resonance data. Eur J Biochem. 1998; 256: 404-410. Rozek A, Friedrich CL, Hancock REW. Structure of the bovine antimicrobial peptide indolicidin bound to dodecylphosphocholine and sodium dodecyl sulfate micelles. Biochemistry. 2000; 39: 15765-15774. Berjeud, J.M., Fremaux, C., Cenatiempo, Y., Simon, L.: US20077238515 (2007). Bibb, M.J., Widdick, D.: US20077166468 (2007). De Vos, W.M., Roelant, J.S., Kuipers, O.P.: US5594103 (1997). Fliss, I., Desbiens, M., Lacroix, L., Tahiri, I., Benech, R. Kheadr, E.: US20017247306 (2001). Hansen, N.: US20036541607 (2003). Not designed: EP1908774 (2008). 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, E.A., Mitsevich, E.V. Mitsevich, I.P.: US20086989370 (2008). Tagg, J.R., Dierksen, K.P., Upton, M.: EP1169340 (2007). Muellner, H., Folger, M., Werner, A., Gierlich, U., Eyer, K., Heinzmann, K., Shaw, N. Wyer, F.: US20070196900 (2007). Van der Weerden NL, Lay FT, Anderson MA. The plant defensin, NaD1, enters the cytoplasm Fusarium Oxysporum Hyphae. J Biol Chem. In press 2008 Attenborough, S., Broekaert, W.F., Osborn, R.W., Ray, J.A., Rees, S.B. Tailor, R.H.: US20006150588 (2000). Broekaert, W.F., Cammue, B.P.A., Osborn, R.W., Rees, S.B.: US5750504 (1998). Broekaert, W.F., Cammue, B.P.A., Osborn, R.W., Rees, S.B., Terras, F.R.G. Vanderleyden, J.: US20016187904 (2001). Duvick, J.P., Rood, T.A., Rao, A.G.: US5905187 (1999). Famodu, O.O., Herrmann, R., Lu, A.L., McCutchen, B.F., Miao, B.F., Miao, G., Presnail, J.K. Weng, Z.: US20077238781 (2007). Manners, J.M., Marcus, J.P., Clifford, K., Green, J.L., Harrison, S.J.: US20056909032 (2005). Manners, J.M., Marcus, J.P., Goulter, K.C., Green, J.L., Bower, N.I.: US20067067624 (2006). Oh, B.J., Ko, M.K., Shin, B.: US20046770750 (2004). Posthuma, G.A., Schaaper, W.M., Sijtsma, L., Van Amerongen, A., Fant, F. Borremans, F.A.: WO01009174 (2001). Reddy, V.S., Hassari, A., Isalm, A.: WO06080032 (2006). Rees, S.B., De Samblanx, G.W., Broekaert, W.F.: US20056864068 (2005). Simmons, C.R., Navarro Acevedo, P.A., Harvell, L., Cahoon, R., McCutchen, B.F., Lu, A.L., Herrmann, R. Wong, J.F.H.: US20056911577 (2005). Weng, Z., Miao, G., Famodu, O.O.: EP1849868 (2008). Ziegenfuss, S., Brinkhaus, F., Greaves, J.: US20006086885 (2000). Carmona MJ, Molina A, Fernández JA, López-Fando JJ, GarcíaOlmedo F. Expression of the alpha-thionin gene from barley in tobacco confers enhanced resistance to bacterial pathogens. Plant J. 1993; 3: 457–462. Vila-Perelló M, Sánchez-Vallet A, García-Olmedo F, Molina A, Andreu D. Synthetic and structural studies on Pyrularia pubera thionin: a single-residue mutation enhances activity against Gramnegative bacteria. FEBS Lett. 2003; 536: 215-219. Broekaert WF, Terras FRG, Cammue BPA, Osborn RW. Plant defensins: novel antimicrobial peptides as components of the host defense system Plant Physiol. 1995; 108: 1353-1358. Lay FT, Anderson MA. Defensins - Components of the innate immune system in plants. Curr Protein Pept Sci. 2005; 6: 85-101. Osborn RW, De Samblanx GW, Thevissen K, et al. Isolation and characterization of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae. FEBS Lett. 1995; 368: 257-262. Segura A, Moreno M, Molina A, García-Olmedo F. Novel defensin subfamily from spinach (Spinacia oleracea). FEBS Lett. 1998; 435: 159-162.

14 Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 [64]

[65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76]

[77] [78]

[79] [80]

[81] [82] [83] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] [100]

[101]

[102] [103] [104]

Van der Weerden NL, Lay FT, Anderson MA. The plant defensin, NaD1, enters the cytoplasm Fusarium Oxysporum Hyphae. J Biol Chem. 2008 (in press); Altier, D.J., Herrmann, R., Lu, A.L., McCutchen, B.F., Presnail, J.K., Weaver, J.L. Wong, J.F.H.: US20056891085 (2005). Altier, D.J., Herrmann, R., Lu, A.L., McCutchen, B.F., Presnail, J.K., Weaver, J.L. Wong, J.F.H.: US20077202214 (2007). Benich, H., Axén, A., Carlsson, A., Engström, A.: US20006063765 (2000). Bulet, P., Hetru, C., Hoffman, R., Sabatier, L.: US20016331522 (2001). Destoumieux, D., Bachere, E., Bulet, P.: US20036642203 (2003). Hultmark, D., Steiner, H., Rasmuson, U., Boman, H.G.: US4355104 (1982). Iwanaga, S., Kawabata, S., Saito, T.: US5861378 (1999). Lai, J.S., Lee, J.H., Callaway, J.E.: US5166321 (1992). Presnail, J.K., Weng, Z., Wong, J.F.H.: EP1146052 (2006). Roch, P., Mitta, G., Hubert, F., Noel, T.: US20056911524 (2005). Schaller, J., Walz, A., Nentwig, W., Kuhn-Nentwig, L.: WO035677 (2003). Mourgues F, Brisset M, Chevreau E. Activity of different antibacterial peptides on Erwinia amylovora growth, and evaluation of the phytotoxicity and stability of cecropins. Plant Science. 1998; 139: 83-91. Jaynes, J., Enright, F.M., White, K.L.: US5962419 (1999). Wachinger M, Kleinschmidt A, Winder A, et al. Antimicrobial peptides melittin and cecropin inhibit replication of human immunodeficiency virus 1 by suppressing viral gene expression. J General Virology. 1998; 79: 731-740. Carlini CR, Grossi-de-Sá MF. Plant toxic proteins with insecticidal properties. A review on their potentialities as bioinsecticides. Toxicon 2002; 40: 1515-1539. Bowdish DME, Davidson DJ, Hancock REW. A re-evaluation of the role of host defense peptides in mammalian immunity. Curr Protein Pept Sci. 2005; 6: 35-51. Bougueleret, L., Chumakov, I.: US20016329340 (2001). Bragger, J.M.: WO07142542 (2007). Day, P.M., Richards, R., Buck, C., Schiller, J.T., Lowy, D.R.: WO06084131 (2006). Donald, C.D.: WO07047512 (2007). Garbe, C., Schittek, B.: US20087348409 (2008). Homan, J., Imboden, M., Riggs, M., Carryn, S., Schaefer, D.A.: US2006147442 (2006). Kim, H., Kang, D., Lee, J.: US20077186795 (2007). 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.: WO07136159 (2007). Lehrer, R.I., Kokryakov, V.N., Harwig, S.S.: US5635594 (1997). Lehrer, R.I., Waring, A.J., Cole, A.M., Hong, T.B.: US20087314858 (2008). McCray, P.B.J., Tack, B.F., Jia, H.P., Schutte, B.C. WO01092309 (2001). Noga, E.J., Silphaduang, U.: US20046753407 (2004). Olsen, H.S., Ruben, S.M.: US20077223840 (2007). Selsted, M.E., Cullor, J.S.: US20016211148 (2001). Selsted, M.E.; US20046696559 (2004). Shokrollah, E., Gerdts, V., Babiuk, L.: WO06002520 (2006). Wehkamp, J., Huang, N., Bevins, C.L., Stange, E.: WO07081486 (2007). Zasloff, M., Bevins, C.L., Diamond, G.: WO007873 (1992). Zasloff, M., Resnick, N.M.: WO018516 (1992). Jing W, Svendsen JS, Vogel HJ. Comparison of NMR structures and model-membrane interactions of 15-residue antimicrobial peptides derived from bovine lactoferricin. Biochem Cell Biol. 2006; 84: 312-326. Gifford JL, Hunter HN, Vogel HJ. Lactoferricin: a lactoferrinderived peptide with antimicrobial, antiviral, antitumor and immunological properties. Cell Mol Life Sci. 2005; 62: 2588-2598. Levy O. Antimicrobial proteins and peptides of blood: templates for novel antimicrobial agents. Blood. 2000; 96: 2664-2672. Scott MG, Yang H, Hancock REW. Biological properties of structurally related -helical cationic antimicrobial peptides. Infect Immun. 1999; 67: 2005-2009. Zasloff M, Martin B, Chen HC. Antimicrobial activity of synthetic magainin peptides and several analogues. Proc Natl Acad Sci USA. 1988; 85: 910-913.

Rizza et al. [105]

[106] [107]

[108] [109] [110] [111] [112] [113] [114] [115] [116] [117] [118] [119] [120] [121] [122]

[123]

[124] [125]

[126]

[127]

[128] [129] [130]

[131] [132]

[133]

Baker MA, Maloy WL, Zasloff M, Jacob LS. Anticancer efficacy of magainin 2 and analogue peptides. Cancer Res. 1993; 53: 30523057. Rinaldi AC, Di Giulio A, Liberi M, et al. Effects of temporins on molecular dynamics and membrane permeabilization in lipid vesicles J Pept Res. 2001; 58: 213-220. Ganz T, Liu L, Valore EV, Oren A. Posttranslational processing and targeting of transgenic human defensin in murine granulocyte, macrophage, fibroblast, and pituitary adenoma cell lines. Blood. 1993; 82: 641-650. Valore EV, Ganz T. Posttranslational processing of defensins in immature human myeloid cells. Blood. 1992; 79: 1538-1544. Nizet V, Gallo RL. Cathelicidins and innate defense against invasive bacterial infection. Scand J Infect Dis. 2003; 35: 670-676. Tomasinsig L, Zanetti M. The cathelicidins - structure, function and evolution. Curr Protein Pept Sci. 2005; 6: 23-34. Lehrer RI, Ganz T. Cathelicidins: a family of endogenous antimicrobial peptides. Curr Opin Hem. 2002; 9: 18-22. Melgarejo, T., Blecha, F., Sang, Y., Ortega, M.T.: WO076162 (2007). Gallo, R.L., Murakami, M.: WO05040192 (2005). Sahl HG, Pag U, Bonness S, et al. Mammalian defensins: structures and mechanism of antibiotic activity. J Leuk Biol. 2005; 77: 466475. Kim, C., Kaufmann, S.H.E., Gajendra, L.: WO06008162 (2006). Ladel, C., Newton, B., Labischinski, H., Brunner, N., Gerdes, C.: WO03105883 (2003). Maury, W., Stapleton, J., Roller, R., Stinski, M., McCray, P.B.J. Tack, B.F.: US20030022829 (2003). Brogden KA. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nature Rev Microb. 2005; 3: 238-250. De Lucca AJ, Walsh TJ. Antifungal peptides: origin, activity, and therapeutic potential. Rev Iberoam Micol. 2000; 17: 116-120. Lee MT, Chen FY, Huang HW. Energetics of pore formation induced by membrane active peptides. Biochemistry. 2004; 43: 3590-3599. Wu YS, Huang HW, Olah GA. Method of oriented circualr dichroism. Biophys J. 1990; 57: 797-806. Yamaguchi S, Hong TB, Waring AJ, Lehrer RI, Hong M. Solidstate NMR investigations of peptide-lipid interaction and orientation of a -sheet antimicrobial peptide, protegrin. Biochemistry. 2002; 41: 9852-9862. Yamaguchi S, Huster D, Waring AJ, et al. Orientation and dynamics of an antimicrobial peptide in the lipid bilayer by solidstate NMR spectroscopy. Biophys J. 2001; 81: 2203-2214. Bechinger B, Zasloff M, Opella SJ. Structure and orientation of the antibiotic peptide magainin in membranes by solid-state nuclear magnetic resonance spectroscopy. Protein Sci. 1993; 2: 2077-2084. Buffy JJ, McCormick MJ, Wi S, et al. Solid-state NMR investigation of the selective perturbation of lipid bilayers by the cyclic antimicrobial peptide RTD-1. Biochemistry. 2004; 43: 98009812. Salgado J, Grage SL, Kondejewski LH, et al. Membrane-bound structure and alignment of the antimicrobial -sheet peptide gramicidin S derived from angular and distance constraints by solid state 19 F-NMR. J Biomol NMR. 2001; 21: 191-208. Balla MS, Bowie JH, Separovic F. Solid-state NMR study of antimicrobial peptides from Australian frogs in phospholipid membranes. Eur Biophys J. 2004; 33: 109-116. He K, Ludtke SJ, Huang HW. Antimicrobial peptide pores in membranes detected by neutron in-plane scattering. Biochemistry. 1995; 34: 15614-15618. Huang HW, Chen FY, Lee MT. Molecular mechanism of peptideinduced pores in membranes. Phys Rev Lett. 2004; 92: 1-4. Matsuzaki K, Murase O, Miyajima K. Kinetics of pore formation by an antimicrobial Peptide, magainin 2, in phospholipid bilayers. Biochemistry. 1995; 34: 12553-12559. Zhao H, Mattila JP, Holopainen JM, Kinnunen PK. Comparison of the membrane association of two antimicrobial peptides, magainin 2 and indolicidin. Biophys J. 2001; 81: 2979-2991. Hancock REW, Rozek A. Role of membranes in the activities of antimicrobial cationic peptides. FEMS Microbiol Lett. 2002; 206: 143-149. Scott MG, Gold MR, Hancock REW. Interaction of cationic peptides with lipoteichoic acid and gram-positive bacteria. Infect Immun. 1999; 67: 6445-6453.

Biomolecules as Host Defense Weapons [134] [135]

[136]

[137] [138]

[139] [140] [141] [142]

[143] [144] [145] [146]

[147]

[148] [149] [150]

[151]

[152]

[153] [154] [155]

[156]

Yeaman MR, Yount NY. Mechanisms of antimicrobial peptide: action and resistance. Pharmacol Rev. 2003; 55: 27-55. Sitaram N, Nagaraj R. Interaction of antimicrobial peptides with biological and model membranes: structural and charge requirements for activity. Biochim Biophys Acta. 1999; 1462: 2954. McCafferty DG, Cudic P, Yu MK, Behenna DC, Kruger R. Synergy and duality in peptide antibiotic mechanisms. Curr Opin Chem Biol. 1999; 3: 672-680. Zhang L, Rozek A, Hancock REW. Interaction of cationic antimicrobial peptides with model membranes. J Biol Chem. 2001; 276: 35714-35722. Yang L, Harroun TA, Weiss TM, Ding L, Huang HW. Barrel-stave model or toroidal model? a case study on melittin pores. Biophys J. 2001; 81: 1475-1485. Laver DR. The barrel-stave model as applied to alamethicin and its analogs reevaluated. Biophys J. 1994; 66: 355-359. Van Kraaij C, De Vos WM, Siezen RJ, Kuipers OP. Lantibiotics: biosynthesis, mode of action and applications. Nat Prod Rep. 1999; 16: 575-587. Huang HW. Elasticity of lipid bilayer interacting with amphiphilic helical peptides. J Phys II. 1995; 5: 1427-1431. Hara T, Kodama H, Kondo M, et al. Effects of peptide dimerization on pore formation: antiparallel disulfide-dimerized magainin 2 analogue. Biopolymers. 2001; 58: 437-446. Zimmerberg J, Kozlov MM. How proteins produce cellular membrane curvature. Nature Rev Mol Cell Biol. 2006; 7: 9-18. Gallucci E, Meleleo D, Micelli S, Picciarelli V. Magainin 2 channel formation in planar lipid membranes: the role of lipid polar groups and ergosterol. Eur Biophys J. 2002; 32: 22-32. Ludtke SJ, He K, Heller WT, et al. Membrane pores induced by magainin. Biochemistry. 1996; 35: 13723-13728. Hallock KJ, Lee DK, Ramamoorthy A. MSI-78, an analogue of the magainin antimicrobial peptides, disrupts lipid bilayer structure via positive curvature strain. Biophys J. 2003; 84: 3052-3060. Bechinger B, Zasloff M, Opella SJ. Structure and dynamics of the antibiotic peptide PGLa in membranes by solution and solid-state nuclear magnetic resonance spectroscopy. Biophys J. 1998; 74: 981-987. Vogel HJ, Jähnig F. The structure of melittin in membranes. Biophys J. 1986; 50: 573-582. Henzler Wildman KA, Lee DK, Ramamoorthy A. Mechanism of lipid bilayer disruption by the human antimicrobial peptide, LL-37. Biochemistry. 2003; 42: 6545-6558. Pouny Y, Rapaport D, Mor A, Nicolas P, Shai Y. Interaction of antimicrobial dermaseptin and its fluorescently labeled analogues with phospholipid membranes. Biochemistry. 1992; 31: 1241612423. Wang W, Smith DK, Moulding K, Chen HM. The dependence of membrane permeability by the antibacterial peptide cecropin B and its analogs, CB-1 and CB-3, on liposomes of different composition. J Biol Chem. 1998; 273: 27438-27448. Ladokhin AS, White SH. 'Detergent-like' permeabilization of anionic lipid vesicles by melittin. Biochim Biophys Acta. 2001; 1514: 253-260. McPhee JB, Hancock REW. Function and therapeutic potential of host defence peptides. J Peptide Sci. 2005; 11: 677-687. Subbalakshmi C, Sitaram N. Mechanism of antimicrobial action of indolicidin. FEMS Microbiol Lett. 1998; 160: 91-96. Chesnokova LS, Slepenkov SV, Witt SN. The insect antimicrobial peptide, L-pyrrhocoricin, binds to and stimulates the ATPase activity of both wild-type and lidless DnaK. FEBS Lett. 2004; 565: 65-69. Kragol G, Lovas S, Varadi G, et al. The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding. Biochemistry. 2001; 40: 30163026.

Recent Patents on DNA & Gene Sequences 2008, Vol. 2, No. 2 [157] [158]

[159]

[160] [161] [162] [163] [164] [165] [166] [167] [168] [169]

[170] [171] [172] [173] [174] [175] [176] [177] [178] [179] [180] [181] [182] [183] [184] [185] [186] [187] [188] [189]

[190] [191]

15

Groisman EA. How bacteria resist killing by host-defense peptides. Trends Microbiol. 1994; 2: 444-448. Belas R, Manos J, Suvanasuthi R. Proteus mirabilis ZapA metalloprotease degrades a broad spectrum of substrates, including antimicrobial peptides. Infect Immun. 2004; 72: 5159-5167. Stumpe S, Schmid R, Stephens DL, Georgiou G, Bakker EP. Identification of OmpT as the protease that hydrolyzes the antimicrobial peptide protamine before it enters growing cells of Escherichia coli. J Bacteriol. 1998; 180: 4002-4006. Campos MA, Vargas MA, Regueiro V, et al. Capsule polysaccharide mediates bacterial resistance to antimicrobial peptides. Infect Immun. 2004; 72: 7107-7114. Nikaido H. Multidrug efflux pumps of Gram-negative bacteria. J Bacteriol. 1996; 178: 5853-5859. Otvos, L.: US20060003938 (2006). Hancock, R.E.W., Karunaratne, N.: US20056906035 (2005). Falla, T.J., Hancock, R.E.W., Gough, M.: US20016191254 (2001). Hahm, K.S., Park, Y., Kwak, J., Park, S.W.: WO06080625 (2006). Blackburn, P., Goldstein, B.P.: WO000694 (1997). Nibbering, P., Hiemstra, P.S., Van den Barselaar, M.T., Pauwels, E.K.J., Feitsma, R.J.: WO054314 (1998). Ulvatne H, Karoliussen S, Stiberg T, Rekdal O, Svendsen JS. Short antibacterial peptides and erythromycin act synergically against Escherichia coli. J Antimicrob Chem. 2001; 48: 203-208. Xiong Y, Yeaman MR, Bayer AS. In vitro antibacterial activities of platelet microbicidal protein and neutrophil defensin against Staphylococcus aureus are influenced by antibiotics differing in mechanism of action. Antimicrob Agents Chemother. 1999; 43: 1111-1117. Li, J., Raynor, C.R., Nation, R.L.: WO06045156 (2006). Hancock, R.E.W., Hilpert, K.: WO06050611 (2006). Roberts, W.K., Selitrennikoff, C.P., Laue, B.E., Potter, S.: US5981844 (1999). Matsuzaki K, Mitani Y, Akada KY, et al. Mechanism of synergism between antimicrobal peptides magainin 2 and PGLa. Biochemistry. 1998; 37: 15144-15153. Zasloff, M.: US5254537 (1993). Yan H, Hancock REW. Synergistic interactions between mammalian antimicrobial defense peptides. Antimicrob Agents Chemother. 2001; 45: 1558-1560. Awasthi, V., Phillips, W.T., Goins, B.A.: WO05079185 (2005). Mastrodonato, M.: WO07144613 (2007). Huang, N., Zhang, D.: WO07081487 (2007). England, R.L.: WO07133373 (2007). Hagie, F.E., Bethell, D., Huang, N.: WO07038623 (2007). De Leeuw, E.P.H., Lu, W.: WO07087557 (2007). Madhyastha, S.: US2007003538 (2007). Madhyasth,a S.: WO05094579 (2005). Hendriks, M.: WO02058752 (2002). Liu Y, Luo J, Xu C, et al. Purification, characterization, and molecular cloning of the gene of a seed-specific antimicrobial protein from Pokeweed. Plant Physiol. 2000; 122: 1015-1024. Heath, R.L., Anderson, M.A.: WO07006079 (2007). Daniell, H.: WO01064927 (2001). Smith, F., Blowers, A.D., Van Eck, J., Sanford, J.: WO006564 (1999). Appendini P, Hotchkiss JH. Review of antimicrobial food packaging. Innovative Food Science & Emerging Technologies. 2002; 3: 113-126. Seydim AC, Sarikus G. Antimicrobial activity of whey protein based edible films incorporated with oregano, rosemary and garlic essential oils. Food Research International. 2006; 39: 639-644. Moïse JA, Han SY, Gudynaite-Savitch L, Johnson DA, Miki BLA. Seed coats: structure, development, composition and biotechnology. In Vitro Cell Dev Bio - Plant. 2005; 41: 620-644.