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Biochemical and Biophysical Research Communications 323 (2004) 721727

Breakthroughs and Views

Avian antimicrobial peptides: the defense role of b-defensins

Haryadi Sugiarto, Pak-Lam Yu*
Biotechnology Group, Institute of Technology and Engineering, College of Sciences, Massey University, Private Bag 11-222, Palmerston North 5301, New Zealand Received 16 August 2004 Available online 11 September 2004

Abstract Avian antimicrobial peptides, classied as b-defensins, have been identied from bloods of chicken, turkey, and ostrich; epithelial cells of chicken and turkey; and king penguin stomach contents. b-Defensins are a family of antimicrobial peptides characterized by six cysteine residues forming b-defensin motifs that are also found in bovine, ovine, pig, and human. These peptides are active against a wide range of microorganisms including Gram-positive and Gram-negative bacteria, fungi, and yeast. Analysis of evolutionary relationships of vertebrate b-defensins showed that there might be a common ancestral gene between avian and other mammalian peptides. This ancient gene may have been passed down and evolved from species older than the oldest living birds, forming a b-defensin-like precursor molecule. This review describes potential applications of these peptides in health care products. 2004 Elsevier Inc. All rights reserved.
Keywords: Antimicrobial peptides; Defensin; b-Defensin

Antimicrobial peptides are relatively small molecules, less than 100 amino acids, which have a broad spectrum of antimicrobial activity. They are cationic molecules as the molecules are rich in histidine, lysine, and arginine. They are also amphipathic, containing hydrophobic and hydrophilic region. They serve as an ancient defense mechanism against pathogenic microorganisms that easily come into contact with the host through the environment. These molecules are considered as part of the innate immune system of all species. Over the past two decades, antimicrobial peptides have been isolated from plants, animals, and human. In animals and human they have been isolated from leukocytes and epithelial cells of skin, gastrointestinal, and respiratory tract ( pag1/htm). Based on the structural size and conformational structure, they are classied into four categories [18]: (1) cysteine-rich amphiphilic b-sheet peptides; (2) amphiphilic a-helical peptides; (3) cysteine-disulde ring

peptides; and (4) linear peptides with one or two predominant amino acids. Due to the large numbers of antimicrobial peptides, this review will focus on avian antimicrobial peptides. They are known as b-defensin molecules, a sub-family of defensins. First, the review describes about what is known about defensins and its classication, and then discusses avian antimicrobial peptides that have been isolated and characterized including range of activity. Lastly, the review discusses evolutionary aspects of this type of antimicrobial peptides and the potential applications in health care products.

Defensins Defensins are cysteine-rich antimicrobial peptides with a triple-stranded b-sheet structure connected with a loop of b-hairpin turn. The main characteristic of defensins molecules is six cysteine residues that form three pairs of disulde bridges. The antimicrobial activities spectra of these defensins include Gram-positive

Corresponding author. E-mail address: (P.-L. Yu).

0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.08.162


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and Gram-negative bacteria, protozoans, some fungi, and enveloped virus, such as human immunodeciency virus (HIV) [4]. There are many defensins that have been isolated from vertebrates that can be classied into three sub-families, a-defensins, b-defensins, and h-defensins (Table 1). Table 1 shows two main defensins, a-defensins and bdefensins, form b-sheet dimers. They dier in the length of residues and the pairing of cysteine linkages that form the disulde bridges. Unlike the other two defensins, hdefensin has a distinctive cyclic structure formed from cyclization of two 9-amino-acid segments of a-defensinlike peptides [22]. This third type of defensin was isolated from rhesus monkey leukocyte [22]. Their precursors were reported to be the products of mutated a-defensin genes with a premature stop codon, resulting in each precursor containing only three cysteine residues [17].

Avian b-defensins Avian b-defensin is classied into the third class among the b-defensin peptides [27]. This classication,

derived by Zhang et al. [27], is based on the length and homology of peptide and gene structures. According to Zhangs classication, the rst class contains a short prepro-sequences (6364 residues) and short introns (less than 1.6 kbp) while the second class contains longer prepro-sequences (6869 residues) than the rst class with more than 6.5 kbp of introns [27]. The source of avian antimicrobial peptides can be separated into two sources: avian heterophil and nonheterophil. Avian heterophil peptides are two chicken heterophil peptides (CHP-1 and -2 [6]); three Gallinacins from chicken (Gal-1, -2, and Gal-1a [13]); three turkey heterophil peptide (THP-1, -2, and -3 [6]); and Ostricacin from ostrich (OSP-1 [25]). The non-heterophil peptides were expressed and characterized from epithelial b-defensins of chicken and turkey [29], and stomach content of king penguin [23]. Table 2 shows the amino acid sequences of these peptides along with two b-defensins isolated from bovine and one from human epithelial cells. As shown in Table 2, the avian peptides share conserved regions with the human and bovine peptides including the three-paired cysteine disulde bridges and a glycine residue.

Table 1 Comparison of vertebrate defensin sub-families [9,18] Structure a-Defensins b-Defensins h-Defensins b-Sheet dimer b-Sheet dimer Cyclic Size (kDa) 3.54 46 2 Residues 2935 3842 18 Cys pairings 16, 24, 35 15, 24, 36 14, 25, 36 Source Human, rabbit, rat, guinea, pig, and mouse Human, bovine, turkey, ostrich, chicken, ovine, pig, and king penguin Rhesus monkey


Ostrich Turkey

Chicken Turkey King Penguin Bovine Human Consensus


Existing antimicrobial peptide sequences that were previously isolated from heterophils (h), epithelial (e), and stomach content (n) of avian species. They are compared with the human epithelial peptide (m), bovine trachea (j), and neutrophil peptide (d).

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Avian heterophil b-defensins Based on the number of homologous residues, the avian heterophil b-defensins can be separated into two sub-classes. The rst sub-class includes THP-1, CHP-1, CHP-2, Gal-1, and Gal-1a. The second sub-class includes THP-2, Gal-2, and Osp-1. The rst sub-class shares a total of 22 identical amino acids, while the second sub-class shares 17 identical residues [6,13]. Between the two sub-classes, they share seven conserved residues, the three-paired cysteine and one glycine, and a proline. Another common characteristic of the second class is that these peptides have two missing amino acid residues after Cys6 residue. Analysis of the ve peptides also showed that there are inter-molecular similarities that can be observed between two Gallinacins (Gal-1 and -2) and two THPs (THP-1 and -2). A study looking at amplication of the two chicken antimicrobial peptides (Gal-1 and Gal-2) and two turkey antimicrobial peptides (THP-1 and THP-2) from their respective bone marrow cDNA was conducted [1]. The study showed that prepro-peptides of Gal-1 and THP-1 contained 65 amino acids with high percentage (85%) of identical amino acid residues. High percentage 93.2% of identical amino acid residues was also shown between the prepro-peptides of Gal-2 and THP-2 [1]. The amplied cDNAs of Gal-2 and THP-2, furthermore, are 68% identical to bovine TAP preregion, whereas Gal-1 and CHP-1 are 56% identical. Gal-2 and THP-2 cDNA also showed 65% identity to bovine LAP and approximately 70% identity to human neutrophil peptide-4, rat neutrophil peptide-1, -2, and -4 [1]. The cDNA amplication study also concluded that there are similarities between Gal-1/CHP-1 and Gal1a/CHP-2 that could be due to genetic heterogeneity or actual cleavage of the terminal amino acid from the mature peptide. The study showed that mature peptide sequences generated from the cDNA amplication of CHP-1 and Gal-1 ended with glycine at the C-terminus, instead of arginine or tryptophan residue (Table 2). Apart from one variation at the C-terminus, which could be due to gene polymorphism, these sequences are almost homologous. Therefore, Brockus et al. [1] proposed to rename one compound as, Gal-1/CHP-1. The other two peptides, Gal-1a and CHP-2, were also renamed as Gal-1a/CHP-2 as they also appeared to be isoforms [1]. These two molecules shared striking similarities with dierentiation in length and two residues, Gal-1a has four residues more than CHP-2 and they have two dierent residues at positions 27 and 30. Avian non-heterophil b-defensins The two epithelial b-defensins, known as Gallinacin-3 (Gal-3) and Gallopavin-1 (GPV-1), were expressed from tracheae of chicken and turkey, respectively [29]. Both

peptides contain 39 amino acid residues. Gal-3 was expressed widely in tongue, Bursa of Fabricius, trachea, skin, esophagus, air sacs, large intestine, and kidney. The expression in trachea was found signicantly aected by Haemophilus paragallinarum [29]. Corresponding cDNA sequences of these two peptides, amplied from the bone marrow, showed 91% identical amino acids up to [29]. These epithelial peptides showed the same number of residues as the two Gallinacins but only 13 identical residues shared identity between them. Gal-3, itself, showed approximately 75% overall identity to the other two Gallinacins, which was mostly marked in their signal sequence and 3 0 untranslated region [29]. Comparison with the two sub-classes of avian heterophils peptides showed that these epithelial peptides share more identical residues to the rst sub-class. In addition to the epithelial peptides, sphenicins are the most recent b-defensins isolated from king penguins stomach contents [23]. There were two isoforms of Sphenicins, namely Sphe-1 and Sphe-2, with 38 amino acid residues and molecular weights of 4482.4 and 4501.4 Da, respectively [23]. The molecular weight dierence was associated with the presence of histidine or arginine residue at position 15 (Table 2). This dierentiation on the isoforms could also be due to gene polymorphism [23]. Comparison to heterophil and epithelial avian b-defensins shows that Sphenicins share more similarities to those of the avian epithelial b-defensins than the avian heterophil b-defensins. The Sphenicins retain 63% similarity to the chicken Gal-3 and 58% similarity to the turkey GPV-1 [23].

Antimicrobial activity of avian b-defensins Most defensins have showed antimicrobial activity against a wide range of pathogens including bacteria and fungi. Avian b-defensins play more important role in the innate defense system because avian heterophil lacks oxidative mechanisms. Oxidative mechanism consists of superoxide ion, hydrogen peroxide, and myeloperoxidase and non-oxidative mechanism consists of few enzymes, cationic proteins, and peptides. Avian heterophils lack superoxide ion and myeloperoxidase, therefore they rely more on the non-oxidative mechanisms that include lysozyme, cationic proteins, and peptides [12]. Previous research work has shown that chicken heterophils inhibited growth of Escherichia coli, Staphylococcus albus, Candida albicans, and Serratia marcescens [2]. At least three cationic proteins or peptides from the heterophil granule extracts were previously reported [3]. Recent research work on avian heterophil b-defensins described earlier has shown activities against a number of microorganisms (Table 3). For comparison, the


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Table 3 Antimicrobial activity of avian heterophil peptides [5,13,23,25] Peptide Gal-1 Gal-1a Gal-2 CHP-1 CHP-2 THP-1 THP-3 Osp-1 Sphe2 BNDBD-1 NP-1 Spectrum of activity Candida albicans, Escherichia coli, and Listeria monocytogenes Escherichia coli and Listeria monocytogenes Candida albicans, Escherichia coli, Bordetella avium, Salmonella enteritidis, Salmonella typhimurium, Campylobacter jejuni, and Mycoplasma gallisepticum Candida albicans, Salmonella enteriditis, and Campylobacter jejuni Escherichia coli and Staphylococcus aureus Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Aspergillus fumigatus (fungi) Escherichia coli and Staphylococcus aureus Escherichia coli and Staphylococcus aureus

Activity of eight avian heterophils and the penguin stomach content b-defensins. The activities of avian epithelial b-defensins have not been reported. These peptides showed activity against a wide range of microorganisms, including Gram-negative, Gram-positive bacteria, yeasts, and fungi.

activities of bovine neutrophil b-defensins (BNBD-1) and rabbit neutrophil peptide (NP-1) are also included. Table 3 shows that most of avian heterophil b-defensins have antimicrobial activity against Gram-positive, Staphylococcus aureus, and Gram-negative bacteria, E. coli. However, only some peptides, such as the chicken and turkey heterophil peptides, have antimicrobial activity against C. albicans [5,13,25]. These antimicrobial activities were measured under optimal conditions, which is low ionic strength and has low concentrations of interfering substances. In contrast to the avian heterophil, synthetic Sphenicin-2 showed antimicrobial activity against a wider range of microorganisms. It has bactericidal activities against all Gram-positive bacteria tested, except against Staphylococcus saprophyticus and bacteriostatic activities against Gram-negative bacteria tested, except E. coli 1106 and Vibrio metsnikovii [23]. Sphenicin-2 also showed inhibition of Aspergillus fumigatus sporulation, but it did not show activity against Candida sp. [23]. The synthetic Sphe-2 also appeared to retain antimicrobial activities at low pH, the conditions of the king penguin stomach contents [23]. It appears that the antimicrobial property of Sphenicins was an important mechanism in preserving the retained food of male king penguin during egg incubation period [23]. The actual mechanisms of how these b-defensins kill microorganisms have not been fully understood. However, many researchers believe that antimicrobial actions of these peptides are based on the two main features of antimicrobial peptides, cationic and amphipathic [7]. Three known models, barrel stave, micellar aggregate, and carpet model, have been developed based on the two main features [19]. In principle, the antimicrobial action is initiated with the binding of b-defensins to bacterial membrane due to electrostatic interaction between the negatively charged

bacterial membrane and the exposed cationic sites of the b-defensins. After the peptide is bound, the negative electric charge of the inner part of the membrane pulls the b-defensin molecule towards the membrane. A dimer is then formed creating a channel between the amino-terminal b-strands of the two b-defensin monomers. The dimer is formed in such a way that the hydrophobic sites of the peptides are facing the inner bacterial membrane and the hydrophilic sites are facing each of the b-defensin monomers [7]. The dimer provides changes in transmembrane potential and causes disruption of membrane permeability that leads to death of cells.

Evolutionary perspective This section provides insights into evolutionary relationship between the avian b-defensins and the other vertebrates b-defensins. It has been shown earlier that apart from the conserved three-paired cysteine residue, b-defensins primary structures are highly varied. Based on amino acid sequence homology, a tree diagram was generated using computer program, Clustal X, to display a relationship between the avian antimicrobial peptides with other b-defensins found in human [15], bovine [21], pig [27], and mouse [16] (Fig. 1). Fig. 1 also shows that the phylogenetic tree agrees with the classication proposed by Zhang et al. [27]. The purication of Ostricacin, Osp-1, from ostrich, a ratite species, could suggest that b-defensin genes have been around for a long time. Ratite is a group of ightless birds found widely in the southern parts of the world (South America, Australia, Africa, New Zealand, and New Guinea). They belong to the oldest Order of living birds, Palaeognathiformes [25]. It was reported that the divergence within ratites might not have

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only b-defensins, but also a-defensins and h-defensins in mammalian lines. h-Defensins could be further evidence of an evolutionary process, since it was recently found with other defensins in the rhesus white cells resembling the human counterparts [22]. This peptide is formed through trimming and ligating of two defensin precursors containing only three cysteine residues. The precursors as described earlier are the products of mutated a-defensin genes with a premature stop codon.

Potential applicationhuman health care products

Fig. 1. Evolutionary tree of existing b-defensins. A phylogeny relationship between avian, bovine, pig, mouse, rhesus monkey, and human b-defensins based on amino acid sequence homology. The bdefensin sequences of avian, bovine, and human are based on the sequence of isolated and puried peptides, while the peptide sequences of pig, mouse, and rhesus monkey are based on successful cDNA expression.

been earlier than 90 million years ago, when the supercontinent Gondwana was broken apart [24]. Since ostrich and mammalians retain b-defensin genes in their genome, the origin of b-defensin genes could be derived from ancient genes that existed at a time before the diversication of the avian and mammalian line had not taken place, approximately more than 150 million years ago [14]. Structural similarity could also be observed when chicken Gal-3 and turkey GPV-1 were compared with a myotoxic peptide found in rattlesnake venom and a peptide from male duck-billed platypus venom [29]. These two compounds have b-defensin motifs. Research on platypus venom shows that the tertiary structure of four peptides contained in the venom resembles that of bovine neutrophil b-defensins-1 (BNBD-1) [17]. Since reptilians can be considered as the oldest vertebrate species, these striking similarities can also indicate another evidence of b-defensins originating from an ancient gene that has gone through evolution. The diversication between vertebrate defensins, adefensins, b-defensins, and h-defensins described earlier could also indicate a common evolutionary origin. Until now, only b-defensins and b-defensin-like peptides have been found in the two old classes of vertebrates, reptile and avian, then it can be suggested that the origin of defensin family comes from b-defensins. Hoover et al. [15] also postulated that the precursor of all defensins was a b-defensin-like molecule from the observation that amino acid sequence and structures of b-defensins resemble those of insect defensins than of mammalian a-defensins. Since insects lines are known to have existed longer than the mammalian lines, it indicates that there is a possibility that the ancestor of b-defensin genes has existed for a longer time. Evolution has produced not

Antimicrobial peptides, being cationic and amphiphilic in nature, have been able to penetrate bacterial wall and defense membrane, causing death of bacterial cells. This mechanism of killing is dierent from that of the classical antibiotics. With the development of antibiotic resistance by some bacteria, there is an urgent need for the development of new therapeutic drugs and antimicrobial peptides can be the answer to the need for new antibiotics [11,28]. Apart from direct killing of pathogenic microorganisms, it has been reported that mammalian and human cationic antimicrobial peptides have other important biological roles, such as adjustment of host inammatory response, chemotactic function to recruit other leukocytes, and as wound healer [9,11,20]. Inammation is host defense reactions due to infections by pathogenic microorganisms. It is usually triggered by the release of bacterial outer membrane component such as lipopolysaccharides (LPS) from Gram-negative bacteria or lipoteichoic acid (LTA) from Gram-positive bacteria [20]. These components induce the production of pro-inammatory cytokines, such as TNF-a, IL-1b, and IL-6. The presence of cationic peptides acts as anti-inammatory agent by binding to the source of inammation LPS or LTA, which leads to the suppression of the LPS or LTA ability to induce the production of pro-inammatory cytokines [11,20]. Anti-inammatory functions of the cationic peptides could be improved by the ability to attract neutrophils, monocytes, macrophage, and T-cells to inammation sites. These leukocytes are commonly found at the inammation sites as part of innate immune response to the presence of pathogens. Hancock et al. [11,20] also suggested that the peptides promote wound healing by recruiting broblast, an essential component in reepithelialization of damaged surface. These biological roles of antimicrobial peptides are illustrated in Fig. 2. In addition to the antimicrobial activities, they also demonstrate synergistic eect with other molecules, such as existing antibiotics, by enhancing the potency of existing antibiotics. Antimicrobial peptides, nisin produced by Lactococcus lactis bacteria and ranalexin


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Fig. 2. Principle functions of defensins in inammatory and defensive reactions against pathogens ( indicates acute inammation response; indicates chronic inammation response) [11,20].

isolated from skin of frog Rana catesbeiana, showed to have activity in vitro in combination with commonly used anti-staphylococcal agents, such as amoxycillin [10]. These peptides appeared to cause degradation of the bacterial cell membrane, facilitating the access of antibiotics to kill the bacterial cell. Other cationic peptides isolated from mammals, indolicidin and protegrin, displayed synergistic eects with host defense molecules such as lactoferrin and lysozyme [20]. Many antimicrobial peptides have shown potential for development into therapeutic drugs and topical products because of their broad range of antimicrobial activities. Up to now, there have been some animal derived antimicrobial peptides, such as pexiganan, protegrin analogue, histatin analogue, human lactorferricin, which have undergone pre-clinical or phase III trials for commercial developments as topical products [26,28]. They are used for antibacterial, antifungal, antiviral, and to combat other infections caused by open wounds. However, there is a safety issue to be investigated before peptide pharmaceutical products can be used. Pexiganan, a magainin analogue, is an antimicrobial peptide, derived from frog skin, and developed for topical use of infected diabetic foot ulcers which have been disapproved by the United States Food Drugs and Administration (FDA) on its Phase III trials due to the high level of its toxicity [26]. The peptide was eective in animal models for infections at very high doses, which are close to the human toxicity level of peptides [26]. Antimicrobial peptides from avian species have shown wide range activity against microorganisms, including antifungal activity against A. fumigatus. Fur-

thermore, avian peptides, similar to mammalian peptides, could possess anti-inammatory functions [8,12]. The mammalian peptides described previously are multifunctional molecules that can act as chemo-attractant to other compounds that inhibit inammatory reaction, signaling molecules that activate immune defense and repair of wound. Therefore, the anti-inammatory function and its high-activity against common pathogenic microorganisms indicate a potential therapeutic capacity of such peptides. These functions of antimicrobial peptides can add value to avian bloods, which are currently waste materials, and the existing emu and ostrich topical products, which are commonly used as moisturizers as well as treatment for muscle aches. The topical products can be used to protect wounds from bacterial or fungal infections, to promote wound healing and as anti-inammatory. Further research works on this area are required to verify the avian peptide functions and for development into useful commercial products.

Conclusion Avian antimicrobial peptides, classied as b-defensins, have been successfully isolated and puried from chicken, ostrich, turkey, and king penguin. These peptides play an important role in avian defense mechanism since avian heterophils lack oxidative mechanisms. In vitro, these peptides have shown to be potent against various microorganisms, including Gram-negative and Gram-positive bacteria as well as fungi. With a wide spectrum of activity and anti-inammatory functions, avian antimicrobial peptides have the potential for development into novel

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human health care products. Comparison between these avian peptides and several bovine and human b-defensins indicated that they share a common ancestral gene. The common ancestral gene could be dierentiated from one precursor, a b-defensin-like molecule. References
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