Anionic Antimicrobial Peptides from Eukaryotic ... - Ingenta Connect

Current Protein and Peptide Science, 2009, 10, 585-606

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Anionic Antimicrobial Peptides from Eukaryotic Organisms Frederick Harris1, Sarah R. Dennison2 and David A. Phoenix3,* 1

School of Forensic and Investigative Science, University of Central Lancashire Preston, PR1 2HE, UK; 2School of Pharmacy and Pharmaceutical Sciences, University of Central Lancashire Preston, PR1 2HE, UK; 3Deputy Vice Chancellor, University of Central Lancashire, Preston, PR1 2HE, UK Abstract: Anionic antimicrobial peptides / proteins (AAMPs) were first reported in the early 1980s and since then, have been established as an important part of the innate immune systems of vertebrates, invertebrates and plants. These peptides are active against bacteria, fungi, viruses and pests such as insects. AAMPs may be induced or expressed constitutively and in some cases, antimicrobial activity appears to be a secondary role for these peptides with other biological activities constituting their primary role. Structural characterization shows AAMPs to generally range in net charge from -1 to -7 and in length from 5 residues to circa 70 residues and for a number of these peptides, post-translational modifications are essential for antimicrobial activity. Membrane interaction appears key to the antimicrobial function of AAMPs and to facilitate these interactions, these peptides generally adopt amphiphilic structures. These architectures vary from the -helical peptides of some amphibians to the cyclic cystine knot structures observed in some plant proteins. Some AAMPs appear to use metal ions to form cationic salt bridges with negatively charged components of microbial membranes, thereby facilitating interaction with their target organisms, but in many cases, the mechanisms underlying the antimicrobial action of these peptides are unclear or have not been elucidated. Here, we present an overview on current research into AAMPs, which suggests that these peptides are an untapped source of putative antimicrobial agents with novel mechanisms of action and possess potential for application in the medical and biotechnological arenas.

Keywords: Amphiphilic helix, antimicrobial peptide, anionic peptide. INTRODUCTION The tissues of living multi-cellular creatures represent a rich source of nutrients from the perspective of the microbe and in response, these creatures have developed a range of defensive measures to microbial challenge. One such measure is the production of antimicrobial peptides / proteins (AMPs), which are evolutionarily conserved components of the innate immune response and constitute the first line of antimicrobial defence for organisms across the eukaryotic kingdom. [1-9]. AMPs are generally located at sites exposed to microbial invasion, such as the epithelia of mammals, amphibians and insects, and show a potent ability to kill a remarkably wide spectrum of microbes and cells, including: parasites [10], tumour cells [11-14], fungi [6,15] and viruses [13,16] along with most Gram-negative and Gram-positive bacteria [17-19]. In the vast majority of cases, AMPs are cationic and kill microbes via mechanisms that predomi-nantly involve interactions between the peptide’s positively charged residues and anionic components of target cell membranes. These interactions can then lead to a range of effects, including membrane permeabilization, depolari-zation, leakage or lysis, resulting in cell death [20-22]. In addition, a number of cationic AMPs appear to target internal anionic cell constituents such as DNA, RNA, or cell wall components whilst others appear to indirectly modulate antimicrobial activity by interacting with other components *Address correspondence to this author at the Deputy Vice Chancellor, University of Central Lancashire, Preston, PR1 2HE, UK; Tel: +44 (0) 1772 892504; Fax: +44 (0) 1772 892936; E-mail: [email protected]

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of the innate immune system [8,23,24]. In general, it is un clear which of these events are primarily responsible for the antimicrobial action of AMPs and multiple target models have been proposed to explain their functionality [22,25,26]. A few cases of intrinsic [7,21,27] and experimentally induced [28,29] microbial resistance to AMPs have been reported. However, these peptides have maintained their efficacy as a eukaryotic weapon against bacterial infection over time. In general, they have not been impaired by microbial responses that lead to acquired resistance and this ability has been ascribed to the relatively non-specific processes involved in the antimicrobial action of AMPs. It has therefore been suggested that this could decrease the likelihood of microbes acquiring resistance to these peptides [18,30]. This could give AMPs a major advantage over conventional antibiotics and, in response, these peptides have been extensively investigated as potential antimicrobial agents. These investigations have shown AMPs to possess several other major advantages over conventional antibiotics. For example, they use microbial killing mechanisms that are extremely rapid and are able to kill pathogens that show multiple-resistance to conventional antibiotics [2,17]. In addition, AMPs can be used in synergistic combination with other members of this peptide family, conventional antibiotics or other antimicrobial agents [31-35]. As a result of these studies, AMPs are increasingly under investigation for commercial development as therapeutic agents with the result that some are in advanced clinical trials and a limited number are currently in use for topical or systemic application [21,30,3640]. Most recently, research has also focused on the use of AMPs as endotoxin-neutralizing compounds and / or agents © 2009 Bentham Science Publishers Ltd.

586 Current Protein and Peptide Science, 2009, Vol. 10, No. 6

to selectively trigger responses from the innate immune system rather than directly kill microbes [5,24,41-44]. Given the great promise shown by cationic AMPs of eukaryotes to function as alternatives to conventional antibiotics, the potential of other groups of AMPs to serve in this capacity has received far less research attention [45,46]. However, since the first reports of cationic AMPs in the early 1980s [47,48], anionic AMPs (AAMPs) have also been described in the literature [49,50]. Over the last decade, these anionic peptides have been increasingly identified in vertebrates, invertebrates and plants, and it has become clear that AAMPs are also an integral and important part of the innate immune system. AAMPs may therefore be a relatively untapped source of putative antimicrobial agents with novel mechanisms of action and in response, a rapidly growing number of these peptides have been recently characterized. Here, we present the first major review of research into eukaryotic AAMPs with a focus on their antimicrobial mechanisms and their potential for application in the medical and biotechnological arenas. Ruminants One of the first reports of anionic ruminant peptides with antimicrobial activity described surfactant-associated anionic peptides (SAAPs, Table 1), which were identified in the pulmonary secretions of sheep and later, cattle [51,52]. These peptides were found to have generally weak activity against a variety of Gram-positive and Gram-negative bacteria, which was enhanced by the presence of Zn2+ to give minimum bactericidal concentrations (MBCs) that were generally > 600 μM [52]. Based on these observations, these latter authors suggested that SAAPs may serve to synergize the action of cationic AMPs and other antimicrobial factors found in the airway surface liquid of ruminants [53]. However, SAAPs were found to be particularly effective against the Gram-negative bacterium, Mannheimia haemolytica, which is strongly associated with respiratory diseases in ruminants. In the presence of Zn2+, these peptides exhibited Table 1.

Harris et al.

MBCs that were < 60 μM when directed against M. haemolytica and were thus of a similar order of magnitude to those reported for cationic AMPs found in ruminant pulmonary secretions [54]. This observation led to the suggestion that SAAPs may be of therapeutic use in treating M. haemolytica for although vaccination against the organism can enhance resistance to infection, these vaccines are not completely effective [53]. Efforts to elucidate the mechanisms underlying the antibacterial activity of SAAPs revealed that they rapidly bound to the surface of M. haemolytica cells, which had been deposited into the lungs of adult sheep, and caused degenerative ultrastructural changes [55]. Previous in vitro studies have shown that in the presence of Zn2+, these peptides induced no morphological changes in M. haemolytica but caused intracellular damage and flocculation of cellular components [52]. Based on these data, it has been suggested that Zn2+ may form a cationic salt bridge between SAAPs and the anionic microbial cell surface. This action would then lead to translocation of these peptides across the cell envelope into the cytoplasm where they could attack internal cellular targets [56]. A number of possible applications for SAAPs in the meat industry have been proposed but their major potential for development appears to be as agents in the treatment of ovine infections, which has been explored in several studies on ruminant respiratory disease [57]. In localisation studies, these peptides were detected in tracheal, hepatic, small intestinal and pulmonary tissue. Within pulmonary tissue, SAAPs were detected in the apical cytoplasm of the bronchial and bronchiolar epithelium, in the cytoplasm of pulmonary endothelial cells and consistent with a role in the innate immune system, in alveolar macrophages [58]. Investigations into calves with acute M. haemolytica infection found that expression levels of these peptides were not increased in comparison to healthy animals, suggesting that SAAPs were constitutively produced [59]. A similar study in sheep suggested that levels of these peptides increased only during chronic M. haemolytica pneumonia and that their

AAMPs from Ruminants

Peptide

Sequence

Reference

SAAP (sheep and cattle)

DDDDDDD

[52]

SAAP (Sheep and cattle)

GDDDDDD

[52]

SAAP (Sheep and cattle)

ADDDDDD

[52]

TAP (Sheep, goat, deer, cow)

VDDDDK

[64]

TAP (Dogfish)

APDDDDK

[64]

TAP (Pig, horse)

TDDDK

[64]

Chromacin (Bovine)

YPGPQAKEDSEGPSQGPASREK

[69]

Peptide B (Bovine)

FAEPLPSEEEGESYSKEVPEMEKRYGGFMRF

[71]

Enkelytin (Bovine)

FAEPLPSEEEGESYSKEVPEMEKRYGGFM

[71]

Kappacin (Bovine, variant A)

MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDSPEVIESPPEINTVQVTSTAV

[84]

Kappacin (Bovine, variant B)

MAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINTVQVTSTAV

[84]

Anionic Antimicrobial Peptides from Eukaryotic Organisms

source was the reparative hyperplastic epithelium. In combination, these results suggested that SAAPs may be important agents in the resolution of infections near epithelial surfaces [60]. Strongly supporting this view, when the efficacy of SAAPs was assessed in a lamb model of acute pneumonia, it was found that a single intratracheal administration reduced pulmonary inflammation and levels of M. haemolytica in infected lung tissue [61]. An economic boost was given to the potential therapeutic use of these peptides by the recent development of a novel bacterial expression system able to produce recombinant SAAPs in very high yield and in an easily recoverable form. Recombinant peptides were found to have equivalent levels of activity to their naturally occurring counterparts and it was proposed that the system could be used for the general mass production of peptide antibiotics [62]. Although studies on M. haemolytica have clearly shown that SAAPs are able to function as AAMPs, in general, these peptides are only weakly antibacterial and it has been proposed that host-defence may not be their primary role [63]. Previous studies have suggested that SAAPs may be derived from precursor proteins [58] and based on their high sequence similarity to the charge-neutralizing propeptides of Group I serine proteases, it was suggested that they may serve a similar function [56]. To further investigate the proposal of these latter authors, the charge-neutralizing activation propeptide of ovine trypsinogen, also a group I serine protease, was assayed for antibacterial activity [64]. These trypsinogen activation peptides (TAPs) are conserved across ruminant species, including sheep, cows and goats, and in the presence of Zn2+, they were found to exhibit comparable antibacterial activity to SAAPs when directed against a range of Gram-positive and Gram-negative organisms. Based on the fact that the post-translational cleavage of TAPs occurs near the surface of epithelial cells in the microenvironment of the mucous layer, it was proposed that these peptides may serve a secondary function by acting as AAMPs on mucosal surfaces [64]. The results of this latter study would seem to support the proposal that antimicrobial activity may be a secondary function of SAAPs [63] and would also appear to be amongst first reports of a protein fragment principally known for other bioactivities functioning in the capacity of an AAMP. It is well established that peptide fragments are post-translationally cleaved from many biologically active proteins such as histones, growth factors [65] and haemoglobins [66] to function as cationic AMPs and augment innate immune responses. Over the last decade, a number of bovine AAMPs have been found to be encrypted within the primary structures of proteins found in the secretory granules of chromaffin cells, which are neuroendocrine cells found in the medulla of the adrenal gland, and in other ganglia of the sympathetic nervous system [67]. Chromogranin A (CGA) is a major glycoprotein (Swiss-Prot accession code: P05059) found in these granules and a naturally cleaved segment prochromacin, which corresponded to residues 79-431 of CGA, was found to be strongly active against both Gram-positive and Gram-negative bacteria [68]. Attempts to localize regions of this segment that were involved in its antibacterial activity led to the identification of three peptides that were designated chromacins (Table 1) and were formed from residues

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173-194 of CGA. This residue sequence endowed chromacins with a net charge of -1, which was further enhanced by post-translational modification: P-chromacin is phosphorylated on tyrosine 173, G-chromacin is O-glycosylated on serine 186 with a trisaccharide moiety, and PG-chromacin possesses both of these architectural modifications. Biological assay showed these peptides to be non-haemolytic and antibacterial in the low M range with bacterial specificity varying according to the post-translational modifications possessed by the peptides. The antimicrobial mechanism of chromacins was not elucidated but the presence of their posttranslational modifications was found to be essential for antibacterial activity and it was speculated that they may be necessary to facilitate the interaction of these AAMPs with bacterial membrane receptors. Interestingly, these AAMPs were only active against Gram-positive bacteria, which is in contrast to prochromacin, and implies that regions of the latter peptide, which lie outside residues 173-194, are required for its activity against Gram-negative bacteria [69,70]. Based on the widespread distribution of CGA in neuroendocrine, endo-crine, nerve and immune cells, and the fact that chromacins are released from these cells in response to stress, it was suggested that these AAMPs are likely to play a role in inflammatory processes by raising an immediate protective barrier against infection thereby potentially playing an important role in the host defense mechanisms [67]. The peptide B / enkelytin family of peptides (Table 2) are well characterized bovine AAMPs (Swiss-Prot accession code: P01211) and were originally identified in the secretory granules of bovine chromaffin cells [71-73] but have since been detected in the plasma and infectious fluids of these animals [74,75]. A number of studies showed that these peptides exhibit high antimicrobial efficacy with with minimum inhibition concentrations (MICs) that were generally < 3 μM when directed against a range of Gram-positive bacteria, including: Micrococcus luteus, Staphylococcus aureus and Bacillus megaterium [72,74]. Later studies on bovine chromaffin cells showed that enkelytin was a truncated form of peptide B and that these peptides were generated by the post-translational processing of proenkephalin A (PEA), a precursor protein of opioid peptides, as shown in Fig. (1). Peptide B was found to be formed by residues 209-239 of PEA whist enkelytin was formed by residues 209-237 of the protein [71,76]. Structural characterization showed that these residue sequences exhibited net charges of -6 and -7 respectively, which were further enhanced by post-translational phosphorylation, and that the antibacterial activity of peptide B / enkelytin depended upon these architectural features. Computer modeling suggested that the active form of these AAMPs was a boomerang-like structure that is essentially two linear -helical segments angularly juxtaposed via a bend induced by a proline residue. In this conformation, glutamic acid residues at positions 228 and 230 of PEA would be brought proximal to phosphorylated serine residues at positions 221 and 223 of the protein, which are essential for the antimicrobial activity of peptide B / enkelytin. It was proposed that the increased levels of negative charge exhibited by these residues due to phosphorylation led to repulsive electrostatic interactions with the glutamic acid residues at positions 228 and 230 of PEA. This action may then promote the opening of the boomerang-like


Table 2.

Harris et al.

Peptide B / Enkelytin from Various Species

[84]Creature

Sequence of enkelytin / peptide B

Swiss-Prot

Bovine

FAEP-LPSEEEGESYSKEVPEMEKRYGGFMRF

P01211

Human

FAEA-LPSDEEGESYSKEVPEMEKRYGGFMRF

P01210

Pig

FADS-LPSDEEGEGYSKEVPEMEKXYGGFMRF

JL0067

Rat

FAES-LPSDEEGESYSKEVPEMEKRYGGFMRF

P04094

Mouse

FAES-LPSDEEGENYSKEVPEIEKRYGGFMRF

P22005

Hamster

FAES-LPSDEEAESYSKEVPEIEKRYGGFMRF

P50175

Guinea pig

FAE-FLPSDEEGESYSKEVPEMEKRYGGFMRF

P47969

Frog

FAE-FLPSDEEGESYSKEVPEMEKRYGGFMRF

P01212

Mussel

FAE-FLPSEEEGESYSKEVPEMERRYGGFMRP

Leech

FAE-FLPSEEEGESYSKEVPEMERRYGGFMRP

Table 2 was adapted from [74] and shows the primary structures of peptide B / enkelytin for a variety of creatures. These sequences are highly conserved, showing homology levels of circa 90%. The Swiss-Prot accession codes of these sequences are given in brackets.

structure possessed by peptide B / enkelytin, thereby facilitating their antibacterial action by allowing the C-terminal amphiphilic -helix to interact with target bacterial membranes. It was also speculated that the phophorylation of peptide B / enkelytin may assist their antibacterial action by permitting the binding of divalent metal ions, thereby promoting conformational change and the adoption of their active form [72,74]. Currently, the precise antimicrobial mechanisms used by peptide B / enkelytin remain unknown but given their co-production with opioid peptides, it has been postulated that these AAMPs participate in a unified neuroimmune response to immediate threat such as bacterial challenge, stress or other stimuli [77,78]. In this response (Fig. 1), PEA processing leads to the liberation of opioid peptides such a Met-Enk, which would participate in the activation of immunocytes and provide a chemotaxic signal to further stimulate immunocyte recruitment. During the time required for this induction and mobilization of the adaptive immune system, peptide B / enkelytin would be released as factors of the innate immune system, providing an immediate counter to invading bacteria, or as a precautionary measure. Degradation of these AAMPs would then generate opioid peptides to augment immunocyte activation / recruitment [78-80]. Kappacins (Table 1) are the first AAMPs to be isolated from bovine milk [81] and more recently have been identified in cheeses made with this milk [82]. These AAMPs are non-glycosylated, phosphorylated forms of caseinomacropeptide (CMP), which are produced by enzymatic cleavage of -casein (Swiss-Prot accession code: P02668), one of the commonest milk proteins [83], to generate a fragment formed by residues 106 to 169. A number of kappacins are known with kappacins A and B (Table 1) by far the best characterised. Both peptides have been shown to exhibit activity against a range of Gram-positive and Gram-negative oral bacteria with MICs in the low μM range. This activity was partially pH-dependent with mildly acidic conditions enhancing up to two-fold the ability of kappacins

Fig. (1). It was adapted from [79] and shows a putative model for the processing of human proenkephalin A in response to microbial challenge. According to this model: the action of prohormone convertase 2 or 3 (SPC2/3) on lysine-arginine (KR) sites in the Cterminal region of the precursor yields a segment containing peptide B. This segment is then cleaved to peptide B, which is then further fragmented by angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) to either enkelytin or Met-Enk RF (MetEnkephalin Arg-Phe). Enkelytin is also degraded by ACE and NEP to Met-Enk. Met-Enk and Met-Enk RF, which then induced chemotaxis.

to inactivate the Gram-positive bacteria, Streptococcus mutans and Actinomycetes naeslundii, both of which are major components of dental plaque. Low pH was also found to enhance significantly the activity of kappacin A against S.


mutans as compared to that of kappacin B against the organism [84,85]. Structural characterisation has shown that both kappacin A and B are strongly anionic and possess sequences with net charges of -7 and -6 respectively that are enhanced in each case by residue phosphorylation. The active region of these AAMPs has been localised to a region corresponding to residues 138-158 of -casein and it was found that phosphorylation of a serine residue at position 149 was essential for their antibacterial activity. These studies also showed that the higher antibacterial activity of kappacin A compared to kappacin B could be ascribed to the single residue difference in the active regions. Kappacin A possesses an aspartic acid residue at position 148 whereas kappacin B displays an alanine residue in the corresponding location [81]. Currently, the antimicrobial mechanisms utilised by kappacins are unclear but based on structural resemblances to enkelytin, it has been speculated that these AAMPs may have similar mechanisms of action. These resemblances include: similar levels of negative charge, the presence of glutamic acid residues and the phosphorylation of serine, which is essential for antibacterial activity. Further resembling enkelytin, kappacins have been shown able to adopt amphiphilic -helical structure in a membrane mimetic environment [86] and the active region of the peptide contains a proline residue. Based on these data, it was suggested that similarly to enkelytin, kappacins may form a proline-kinked amphiphilic -helix. Moreover, it has been shown that in a membrane mimetic environment with divalent metal ions present, kappacins undergo a conformational change to adopt a more rigid structure [84]. It has previously been suggested that divalent metal ion binding may lead to changes in the conformation of enkelytin [74]. The presence of divalent metal ions was also found to enhance the activity of kappacin and it has previously been shown that these peptides are strongly membranolytic at acidic pH, permeabilising liposomes at levels comparable to those of its antibacterial activity under these acidic pH conditions. In combination, these data led to the suggestion that divalent cations may facilitate the interaction of kappacin with the bacterial membrane and / or its ability to aggregate in the membrane to form anionic pores, thus increasing its permeability to cations. Under acid conditions, this action could facilitate the influx of hydrogen ions thereby lowering intracellular pH and contributing to the antibacterial activity of the peptide [84,85]. The ability of kappacin to kill oral bacteria suggested the potential to act as a therapeutic agent in the fight against dental caries and periodontal diseases. However, the major causative factor in both diseases is dental plaque, which consists of a diverse community of oral bacteria that exists on the tooth surface as a biofilm, and it is well established that these biofilms are far more resistant to antibiotics and cationic AMPs than planktonic bacteria [87]. In response, kappacin / Zn2+ preparations were tested and found to be highly effective against a variety of mono and polybacterial biofilms under conditions mimicking the oral cavity. For example, it was found that this preparation reduced viable cell numbers of an S. mutans biofilm by > 90% with no sign of recovery after 15 days. In contrast, although chlorhexi-


589

dine, which is currently considered the most effective oral care biocide [88,89], showed a comparable bactericidal effect, viable cell numbers rapidly recovered after treatment with this agent. It was suggested that the efficacy of kappacins against these oral biofilms may partly reside in the virtual absence of positively charged residues from these AAMPs. It is generally accepted that the efficacy of cationic AMPs against microbial biofilms is reduced by the binding of lysine and arginine residues in these peptides to anionic components of the biofilm matrix, thus inhibiting their ability to interact with target microbes. Moreover, cleavage sites provided by the overrepresentation of lysine and arginine residues in cationic AMPs render these peptides susceptible to inactivation by bacterial proteinases associated with dental plaque. Based on these combined data, it was suggested that kappacins may have the potential to act as novel antibacterial additives to food, toothpaste and mouthwashes [84,85]. Homo Sapiens Earlier studies have reported the occurrence of AAMPlike peptides in human bronchoalveolar lavage fluid (BAFL), which required the presence of Zn2+ for antimicrobial activity [90]. These results led to investigation of the expression pattern of AAMPs in the BAFL of individuals with cystic fibrosis (CF) [91], which is characterised by a progressive loss of pulmonary function, resulting from a cycle of inflammation and bacterial infections [92]. Using antibodies raised against the ovine defence peptides, (Table 1) these latter investigations found that AAMP-like molecules were present in the BAFL of healthy humans but occurred at significantly lower levels in patients with cystic fibrosis. Similar analyses of pulmonary tissue found differences in the levels of these AAMP-like molecules in the apical cytoplasm of the bronchial and bronchiolar epithelium and in the alveolar epithelium of the subjects tested. It was concluded that these molecules are present in healthy human respiratory tracts and differ in concentration and location of expression in patients with and without CF. Based on these data, it was suggested that a physiologic deficiency in these AAMP-like molecules may predispose an individual with CF to respiratory infections [91] but no further investigations into this suggestion appear to have been conducted. The observed distribution pattern of these AAMP-like molecules contrasts to that found with cationic AMPs and other antimicrobial molecules, which occur at similar levels in individuals with and without CF. Moreover cationic AMPs and other antimicrobial molecules are generally ineffective in the high salt conditions encountered on the apical side of lung epithelial cells in CF related infections [93]. Given the lethal nature and widespread occurrence of CF along with the recalcitrance of its associated lung infections to treatment with conventional antibiotics [94], it may be fruitful to characterise further the AAMP-like molecules described in the above studies [91]. Currently, a number of AMPs exogenous to the lung are under consideration for treating CF related lung infections but none appear to be currently at the stage of clinical trials [21]. A variety of AAMPs have been found to be encrypted within the primary structures of proteins identified in the plasma and cells of human blood. Peptides corresponding to residues 21-53 of the lipid transport protein, apolipoprotein


CIII (Swiss-Prot accession code: P02656) and residues 21-50 of the fibrinogen -chain (Swiss-Prot accession code: P02671), which is involved in blood coagulation (Table 3) have been identified in human plasma. These sequences carried a net charge of -2 and both peptides were reported to be antibacterial but as yet neither appear to have been further characterized [45]. Interestingly, the latter peptide included a segment corresponding to fibrinopeptide A (residues 21-35 of the fibrinogen -chain), which possessed a net charge of 3 and was also shown to function as an AAMP (Table 3) in human platelets, apparently after proteolytic cleavage from fibrinogen [95]. Two further AAMPs (Table 3) with a net charge of -2 were identified in these platelets, which were fibrinopeptide B (residues 31-44 of the fibrinogen -chain; SwissProt accession code: P02675) and thymosin-4 (SwissProt accession code: P62328). Each of these AAMPs identified in platelets was active in the low μM range against pathogens that have a propensity to enter the bloodstream, including S. aureus, the Gram-negative organism, Escherichia coli, and the fungi, Candida albicans, and Cryptococcus neoformans. However, the pH optima for this activity varied with those of fibrinopeptides A and B enhanced by acidic conditions and that of thymosin-4 by alkaline conditions. Based on these observations, it was suggested that the antimicrobial activities of these AAMPs served to complement and synergize those of cationic AMPs identified in platelets, thereby providing these cells with broad range efficacy against blood borne pathogens. Taken with other data, this study was one of the first to suggest a direct antimicrobial role for platelets in the response to trauma or the mediation of inflammation and that this antimicrobial role included the release of AAMPs [95]. Since these latter studies, there appears to have been relatively little research aimed at further characterization of platelet AAMPs. Fibrinogen peptide A was found to be ineffective against strains of the Grampositive bacteria, Lactobacillus rhamnosus and Lactobacillus paracasei, when testing the infectivity of these organisms in a rat model of experimental endocarditis [96] but based partly on its antimicrobial and anti-inflammatory properties, thymosin-4 was found to exhibit significant potential for use as an aid in oral healthcare [97]. Sequence analysis of human proenkephalin (PEA) showed that peptide B / enkelytin were encrypted within the primary structure of the protein and exhibited high levels of homology to their bovine counterparts (Table 2). These human peptides have been detected in the secretions of polymorphic neutrophils and, in response to a bacterial challenge, they were quickly released at the low μM levels associated with their antibacterial action [74]. A recent study on patients undergoing coronary artery bypass grafting showed that peptide B and enkelytin were initially present at low levels in plasma but that these levels quickly rose just after skin incision. These AAMPs showed a similar range of antibacterial activity to their bovine counterparts, specifically targeting Gram-positive organisms with MICs in the low μM range. Moreover, human peptide B and enkelytin were metabolized in vivo to opioid peptides with granulocyte chemotactic activity [98] further supporting the view that the nervous and immune systems present a unified response to immediate threat (Fig. (1); [78]).

Harris et al.

Dermcidin (DCD) is one of the most studied human AAMPs and its gene sequence was first identified when subtractive hybridization techniques were used to identify transformation-related genes in malignant melanoma. This gene was found to encode a full length protein of 110 residues [99] and encrypted within the sequence of this protein were a number of peptides, which are liberated by post-translational cleavage, including Y-P30, PIF and DSEP [100-102]. DCD and these daughter peptides appear to perform a variety of biological functions ([100,103-108] ranging from the regulation of genes for breast cancer cell metabolism, proliferation and survival [109] to aiding the regulation of placental function [110]. However, a major research focus over the last decade has been on the role of DCD and its derivative peptides as AAMPs [111-116]. The presence of DCD in neutrophil granules of a variety of mammals has been recently demonstrated [117-119] but the protein was first shown to play a role in the innate immune system of humans over five years ago [120]. It has been demonstrated that DCD was constitutively expressed in the dark mucous cells of the eccrine sweat glands, secreted into sweat and transported to the epidermal surface [120,121]. However, a very recent study on burned human skin tissue has questioned the constitutive expression of DCD under inflammatory conditions and suggested that the regulation of the protein’s expression requires further investigation [122]. A number of studies have shown that DCD is proteolytically processed in sweat to produce DCD-1 (residues 63109) and DCD-1L (residues 63-110), which also function as AAMPs (Table 3). Further processing of DCD-1 and DCD1L by a number of sweat-borne proteases including: cathepsin D, a 1,10-phenanthroline-sensitive carboxypeptidase and an as yet unknown endopeptidase, yielded a range of truncated peptides. These truncated peptides included AAMPs with net charges up to –2 (Table 3) and other AMPs with net charges up to +2, that together exhibited a spectrum of antimicrobial action, which varied with the individual [120,123-126]. The antimicrobial activity of these peptides occurred in the low μM range and was unaffected by low pH and elevated NaCl levels, conditions that are characteristic of human sweat. Based on these results, it was suggested that DCD and its daughter peptides play a major role in the innate immune responses of the skin [120,121]. Strong support for this suggestion came from studies on atopic dermatitis (AD), which is manifested by recurrent bacterial or viral skin infections [127,128]. Compared to healthy individuals, sufferers of AD were found to have reduced amounts of DCD and its derivative peptides in their sweat and an impaired ability to kill bacteria on the skin surface [129,130]. DCD and its derivative peptides are highly effective against a range of bacteria and fungi under conditions that mimic human sweat. DCD was found to inactivate E. coli, S. aureus and [131], and Staphylococcus epidermidis. This latter organism is the predominant Gram-positive microbe on human skin and a major nocosomal pathogen, frequently forming highly recalcitrant biofilms in medical devices such as catheters [132,133]. Both DCD-1L and DCD-1 were found to exhibit antimicrobial activity against a wide range of nocosomal pathogens, including Gram-positive bacteria


Table 3.


591

AAMPs from Homo sapiens

Peptide Fibrinogen -chain21-50

Sequence

Reference

DSGEGDFLAEGGGVRGPRVVERHQSACKDS

[45]

SEAEDASLLSFMQGYMKHATKTAKDFTALSSVQES

[45]

Fibrinopeptide A

DSGEGDFLAEGGGVR

[95]

Fibrinopeptide B

QGVNDNEEGFFSAR

[95]

SDKPDMAEIEKFDKSKLKKTETQEKNPLP SKETIEQEKQAGES

[95]

DLLPPRTPPYQEPASDLKVVDCRRSEGFCQEYCNYMETQVGYCSKKKDACCLH

[141]

SSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDSVL

[120]

DCD-1

SSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDSV

[120]

SSL-46

SSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDS

[123]

SSL-45

SSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLD

[123]

LEK-45

LEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDSVL

[123]

LEK-44

LEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDSV

[123]

LEK-43

LEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDS

[123]

LEK-42

LEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLD

[123]

LEK-41

LEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVL

[123]

Apo CIII21-53

Thymosin-4 HE2C DCD-1L

such as Enterococcus faecalis, Listeria monocytogenes, methicillin-resistant S. aureus, and rifampicin and osoniazardresistant Mycobacterium tuberculosis, Gram-negative bacteria such as E. coli, Salmonella typhimurium, Pseudomonas putida, and Pseudomonas aeruginosa, and fungi such as C. albicans [111,115,120,134]. Antimicrobial activity has also been observed for shorter DCD fragments such as SSL-46 [130] and SSL-25, which was more effective against Gramnegative organisms than DCD-1L itself [123]. Most recently, a few cases of bacterial resistance to DCD have been described. Both S. epidermidis and S. aureus were reported to raise their extracellular proteolytic activity in response to challenge by DCD, resulting in degradation of the peptide [114]. P. aeruginosa also shows resistance to the action of DCD and its daughter peptides but this was found not to be mediated by proteolytic degradation under physiological conditions and the basis of this resistance is as yet unclear [111]. S. epidermidis has also been reported to generate resistance to DCD by producing the exopolysacharide, intercellular adhesin, which is a cationic polymer and appears to sequester the protein, thereby preventing it from reaching its cellular target(s) [132]. Structure / function studies have shown that DCD possesses no significant levels of defined secondary structure in aqueous solution but acquires -helix and -sheet architecture in the presence membrane-mimicking environments and it was suggested that this transition to more ordered structures may be relevant to some biological functions of the protein [135]. The structure / function relationships underlying the antimicrobial action of DCD derived peptides has been addressed by a number of studies. Peptides derived from DCD have been shown to adopt only low levels of am-

phiphilic -helical structure in the presence of membrane mimics [115,136]. Moreover, this latter study found that the antimicrobial activity of DCD-derived peptides showed no correlation with their levels of secondary structure or net charge. Contrasting with most cationic AMPs, DCD-derived peptides showed no evidence of an ability to perturb membranes and a model was proposed whereby these peptides oligomerise and bind to the bacterial membrane [136]. The antimicrobial activity of these peptides then proceeds via receptor-type interactions with targets on the bacterial membrane surface, possibly also involving internalisation and targeting of sites within the bacterial cell as previously described for a growing number of cationic AMPs [25]. A spectrum of -defensins, which are generally cationic AMPs with amphiphilic structures, have been identified in the human epydidymis, an organ actively involved in the post-testicular sperm maturation process. These peptides play an important role in local protection from infections as adaptive immunity is largely suppressed in the male genital tract [137,138]. In previous studies it was predicted that an anionic -defensin type peptide, HE2C (Swiss-Prot accession code: Q08648), would be expressed in the human epydidymis [139,140] and more recently, the peptide was cloned and characterised [141]. These studies showed that HE2C (Table 3) was secreted into human epididymal fluid and became part of the ejaculate. However, sequence analysis revealed that the peptide was an atypical -defensin in that it carried a net charge of -1 and did not appear to be an amphiphilic molecule. Moreover, in further contrast to most cationic -defensins, the peptide showed no activity against E. coli and S. aureus and it was suggested that HE2C may serve some function other than innate defence. Nonetheless,


the peptide was found to bind strongly to the surface of these latter organisms by some undetermined mechanism and it was speculated that HE2C may be active against other organisms. It has been suggested that HE2C should be further investigated, given the current interest in developing human defensins as contraceptives, topical biocides against sexually transmitted diseases and antiviral agents [142,143]. Amphibians The amphibian skin surface is a rich source of cationic AMPs with the African clawed frog, Xenopus laevis, producing magainins, which, along with a host of derivatives, are to date the best studied AMPs for therapeutic purposes [40]. In contrast, the only anionic peptide with antimicrobial activity reported for X. laevis appears to be the activation peptide from the PYL protein of the organism (Table 4). This peptide exhibited a net charge of -5 and showed high levels of homology to ovine TAPs (Table 1). Similarly to these latter peptides, the PYL activation peptide was found to have generally weak activity against a variety of Gram-positive and Gram-negative bacteria, which was enhanced by the presence of Zn2+ to give MICs that were generally > 250 μM [64]. Based on these observations, it was suggested that hostdefence may not be the primary role of the peptide but as a secondary role, it may complement the antimicrobial barrier presented by cationic AMPs present in the skin and mucosal epithelium of X. laevis [64]. In addition, peptide B / enkelytin homologues have been shown to be present within the proenkephalin A sequence of X. laevis (Swiss-Prot accession code: P01212) along with that of other frogs (Swiss-Prot accession code: Q5FW03), toads (Swiss-Prot accession codes: Q9I817, and Q5Y4B6), newts (Swiss-Prot accession code: Q5PZ00) and salamanders (Swiss-Prot accession codes: Q6YIR3, Q6YIR4) although no major experimental investigations into this observation appear to have been conducted. A well characterized family of cationic amphibian AMPs is the dermaseptins, which derive from South American frogs of the genus, Phyllomedua. Closely related to the latter frogs are Agalychnis annae and Pachymedusa dacnicolor and the use of cDNA derived from the skin of these amphibians to identify dermaseptins and related peptides, led to the description of three AAMPs. AA-2-5 (Table 4), which carried a net charge of -1 (Swiss-Prot accession code: O93222) was derived from the former organism whilst PD3-6 and PD-3-7 (Table 4), which carried net charges of -1 and -2 respectively (Swiss-Prot accession codes: O93454 and O93455), were derived from the latter organism. These peptides were not investigated for antimicrobial activity but it was predicted that they would form membrane interactive helices with similar function and properties to dermaseptins [144]. These AAMPs may be of therapeutic interest as dermaseptins are amongst the few vertebrate peptides that are able to inactivate viruses such as herpes simplex virus (HSV) and human immunodeficiency virus (HIV) along with fungal pathogens that are often associated with these viral infections. Based on this ability, it has been suggested that dermaseptins may be of use in the treatment of sexually transmitted diseases and infections that follow the immunodeficiency syndrome or the use of immunosuppressive agents [145-147].

Harris et al.

The temporins are also well established cationic amphibian AMPs that were initially isolated from the European frog, Rana temporia [148]. Recently, an anionic temporin (Swiss-Prot accession code: P83007) was isolated from the skin secretions of the Japanese brown frog, Rana japonica [149]. Designated temporin-1Ja (Table 4), this peptide carried a net charge of -1 and showed moderate activity against E. coli and S. aureus with MICs > 100 μM. The peptide was not further characterised and its physiological role is unknown. However, previous studies have shown that cationic temporins with comparable antibacterial activity to temporin-1Ja can synergise with other isoforms of these AMPs produced within the same frog specimen to neutralize endotoxins. Based on these observations, it has been proposed that temporin-1Ja may be of use in developing novel antisepsis drugs [33,150]. The best characterized amphibian AAMPs so far reported appear to be produced by the Bombina genus. Bombinins H are antimicrobial peptides, which were originally isolated from skin secretions of the yellow-bellied toad, Bombina variegate. This family of antimicrobial agents is unusual in that some members are post-translationally modified to produce pairs of epimeric peptides where one is an all L-isomer and the other possesses the corresponding D-amino acid in the second position of its primary structure [151]. Genes coding for anionic bombinin H peptides (Swiss-Prot accession code: P29002) were identified in Bombina orientalis and these peptides were synthesized as the all L-isomer, GH1L, and GH-1D, which possessed D-alloisoleucine at sequence position 2 (Table 4). Contrasting to other bombinin H peptides, these latter epimers appeared not to adopt -helical structure, showing no defined conformation. In further contrast, they exhibited high levels of haemolysis and were inactive against yeasts, Gram-positive bacteria and Gramnegative bacteria with the exception of Aeromonas hydrophila where an MIC of 200 M was observed for both peptides. This latter Gram-negative organism is one of the most common bacterial strains found in amphibia and it was suggested that GH-1L and GH-1 D may act in concert with other bombinin H peptides to present a broad spectrum of antimicrobial activity for effective modulation of the natural flora of Bombina species [152]. In more recent studies on Bombina maxima, the Chinese red belly toad, use of a cDNA library derived from skin of the amphibian led to the identification of maximin H5 [153]. This peptide (Table 4) possessed a net charge of -3 and was found to exhibit weak activity against S. aureus with an MIC of 800 μM but showed no activity against Gram-negative bacteria or fungal strains. These studies established that in contrast to some AAMPs, such as those of ovine origins described above (Table 1), the antimicrobial action of maximin H5 did not require the presence of divalent metal ions. The peptide also showed the potential to form a membrane interactive -helix but no investigation into the role of this structure in the antimicrobial mechanism of maximin H5 appear to have been conducted [153]. Most recently, a synthetic homologue of maximin H5, AP1, which carried a net charge of -2, was found to exhibit weak activity against both E. coli and S. aureus with MICs of circa 2 mM. However, although this activity appeared to involve membrane interaction via the adoption of -helical structure in both cases, a different


Table 4.


593

AAMPs from Amphibians

AAMP PYL activation peptide

Sequence

Reference

ADADDDDDK-OH

[64]

AA-2-5

GLVSGLLNTAGGLLGDLLGSLGSLSG

[144]

PD-3-6

GVVTDLLNTAGGLLGNLVGSLSG

[144]

PD-3-7

LLGDLLGQTSKLVNDLTDTVGSIV

[144]

ILPLVGNLLNDLL-NH 2

[149]

GH-1L

IIGPVLGLVGKPLESLLE-NH2

[152]

GH-1D

IIGDPVLGLVGKPLESLLE-NH2

[152]

ILGPVLGLVSDTLDDVLGIL-NH2

[153]

GEQGALAQFGEWL

[155]

Temporin-1Ja

Maximin H5 AP1

bacterial killing mechanism was found to be utilized by the peptide for each organism. These results are important to the current drive to design new antimicrobial compounds with novel mechanisms of action in that they clearly demonstrated that it is possible for an AAMP to utilize more than one mechanism of antimicrobial action depending upon its target organism. Strategies to design antimicrobial compounds must therefore accommodate the fact that both the structural characteristics of these compounds and the composition of their target membrane are important determinants in the efficacy of their action against target organisms [154,155]. Rodents AAMP-like peptides have been described in the bronchoalveolar lavage fluid of mice [50] and peptide B / enkelytin homologues have been shown to be present within the proenkephalin A (PEA) sequences of mice, hamsters, guinea pigs, rats and other rodents (Table 2) although these peptides appear not to have been further characterised. Earlier studies on rat brains detected immunoreactive forms of peptide B at high levels in the hypothalamus and the striatum but at lower levels in the midbrain and medulla-pons of the animal [156]. More recent studies on rat spleen investigated the expression of proprotein convertases (PCs) that process PEA to enkelytin in immune cells. These studies demonstrated that these PCs were expressed under basal conditions in both macrophages and lymphocytes. However, in the spleen of rats that had been treated with lipopolysaccharide (LPS), thereby mimicking bacterial infection, the expression levels of these PCs were increased, and were accompanied by the induction of PEA expression and processing to yield enkelytin and opioid peptides. These studies confirmed that the expression of PCs involved in the production of enkelytin and peptide B was not restricted to neurons and endocrine cells of rats and that these enzymes played significant roles in the innate and adaptive immune systems of these animals [157]. Recent studies also suggested that AAMPs derived from DCD may be present in these rodents when the mRNA of this protein (Swiss-Prot accession code: Q71DI1) was demonstrated in the footpad sweat glands of juvenile rats [158].

AAMPs FROM INVERTEBRATES Crustaceans Relatively few AMPs have been derived from crustaceans with penaeidins from shrimps amongst the best characterised [159]. However, recent studies on the shrimps, Penaeus stylirostris and Penaeus vannamei, reported the presence of AAMPs in the plasma of these organisms [160]. Sequence alignment showed that these peptides, PvHCt from P. vannamei and PsHCt1 and PsHC2 from P. stylirostris (Table 5), were derived from the C-terminal region of haemocyanin (Swiss-Prot accession codes: Q26180; Q9NFY6), the respiratory protein of crustacean plasma. The mechanism responsible for this processing of haemocyanin was not determined but was predicted to involve limited proteolysis of the protein. When tested against a range of microbes, it was found that PvHCt, PsHCt1 and PsHC2 possessed no antibacterial activity but were highly active against Fusarium oxysporum, a fungal shrimp pathogen. Subsequent assay of synthetic PvHCt showed the peptide to have potent broad range antifungal activity with MICs that were < 15 M when directed against F. oxysporum, Fusarium culmorum, Alternaria brassicola, Nectria hematococca, Neurospora crassa and Tricoderma viridae. The antifungal mechanism of these peptides was not investigated but sequence alignments showed that the primary structure of these peptides did not include the copper-binding site involved in the biological activities of haemocyanin. This observation appeared to exclude the possibility that the antifungal activities observed for PvHCt, PsHCt1 and PsHC2 involved the presence of heavy metals or binding sites for divalent ions, as is the case for other AAMPs, such as ovine SAAPs, described above (Table 1). Founded on these studies, it was suggested that haemocyanin may play a role in crustacean innate immunity by serving as a substrate for the generation of antifungal peptides, which could be rapidly produced in response to microbial infection. Moreover, given that shrimp farming is a global economic activity, it was proposed that the insight provided by this study could help in the design of more efficient strategies for disease control, since shrimps are increasingly affected by infectious diseases, particularly those of fungal origin [160].


Harris et al.

Several putative AAMPs have been reported from Scylla serrata, the Indian mud crab, which included: an unnamed antimicrobial protein (Swiss-Prot accession code: A1YV58) and scygonadin 2 (Swiss-Prot accession codes A9NJG5). Whilst these peptides have only been detected at the transcript level, they show high levels of homology to scygonadin (Table 5), which is a novel antibacterial peptide recently isolated from the seminal plasma of S. serrata (SwissProt accession code: Q5D710). These latter studies showed that scygonadin possessed potent activity against M. luteus, inhibiting the organism at circa 10 μM [161]. More recently, sequence analysis of the cloned scygonadin gene predicted a propeptide with a signal sequence of 24 residues, indicative of a secreted molecule, and a mature peptide of 102 residues with an overall amphiphilic structure that possessed a net charge of -1. Mechanisms underlying the antimicrobial activity of scygonadin were not investigated but it was speculated that this activity may involve a number of regions within the mature peptide, including a cationic N-terminal segment defined by residues 1-13 and an anionic C-terminal segment centred on residues 93-97 [162]. Later work showed that the scygonadin gene was not expressed in the early larval stages of S. serrata or in females but specifically in the ejaculatory vesicle of the male crab’s reproductive apparatus [163]. Based on these investigations, it was proposed that the peptide played a role in protecting the male crab reproductive tract from invading pathogenic micro-organisms, thereby maintaining a sterile environment for the fertilization process. Open questions, which were proposed for future work [162] included: whether scygonadin expression was induced in response to microbial challenge or by mating, and whether transfer of the peptide from male to female in the seminal fluid occurred during the mating process for the purpose of protecting the female and the sperm from mating-introduced bacteria. Annelids and Molluscs Annelids have developed efficient immunodefence strategies against microbes living in water or soil that are ingested during feeding or introduced into the body after injury. The most intensively studied of these immune defence systems are those of the Oligochaeta (Earthworms) and Hirudinea (leeches), which comprises coelomocytes and other cell types that float in the fluid of the coelomic cavity and secrete antimicrobial agents into this fluid [164]. Recently, an AAMP named theromyzin (Swiss-Prot accession number: Q6T6C1) was isolated from the coelomic liquid of the leech Thermyzon tessulatum along with a cationic AMP Table 5.

named theromacin [165]. Each of these peptides possessed a putative signal sequence, consistent with secreted proteins, and only exhibited activity towards Gram-positive bacteria. Theromyzin (Table 6) carried a net charge of -4 and showed potent activity against M. luteus with an MIC < 1 μM. Although the precise antibacterial mechanism(s) used by the peptide was not determined, it was observed that the Nterminal region of theromyzin was rich in histidine and aspartate residues. The peptide thus showed some structural resemblance to the aspartate rich ovine SAAPs described above (Table 1) and the histidine rich, cationic AMPs, human histatins [166,167]. On this premise, it was suggested that the N-terminal region theromyzin could be responsible for the antibacterial activity of the peptide and may require interaction with a cofactor such as Zn2+ for this activity. Localization of theromyzin showed that it was expressed exclusively in large fat cells of the annelid and rapidly released into the coelomic fluid in response to septic injury or bacterial challenge, suggesting that the peptide may exert its antimicrobial activities through systemic action. Theromyzin was also detected in intestinal epithelial cells, suggesting a role in epithelial defence. Taken together, these data suggested a scheme for the role of the peptide in the immune system of T. tessulatum whereby septic injury would provoke the production of mucous, which would entrap invading bacteria in the external environment of the leech. These trapped bacteria would then be attacked by theromyzin and theromacin present in the mucous. Concomitantly, injury would induce the expression of these peptides and their secretion into the coelomic fluid of the animal to exert their systemic antibacterial action. In conjunction with the phagocytic action of coelomocytes, this would lead to the killing of bacteria in the body fluid [164,165]. More recent studies have suggested that theromyzin and theromacin participate in the neuroimmune response of T. tessulatum. Following trauma, the central nervous system (CNS) of the animal has a strong capacity to regenerate and restore normal function. It was found that microbial components induce the transcription of theromyzin and theromacin genes in microglial cells, which are the main resident immunological cells of the CNS, with the products of these genes rapidly accumulating at sites in the CNS undergoing regeneration. These studies also showed that theromyzin and theromacin were synthesized in neurons and that in addition to exerting antimicrobial properties, both peptides acted as promoters of the regenerative process in the leech CNS. It was concluded the leech CNS would make a good model system for studying the implication of immune molecules in neural repair [168].

AAMPs from Crustaceans

Peptide

Sequence

Reference

PsHCt1

VTDGDADSAVPNLHENTEYNHYGSHGVYPDK

[160]

PsHCt2

LVVAVTDGDADSAVPNLHENTEYNHYGSHGVY

[160]

PvHCt

FEDLPNFGHIQLKVFNHGEHIHH

[160]

Scygonadin

GQALNKLMPKIVSAIIYMVGQPNAGVTFLGHQCLVESTRQPDGFYTAKMSCASWTHDNPIVGEGRSRVELEALKGSITNFVQTASNYKKFTIDEVEDWIASY

[162]


Table 6.


595

AAMPs from Annelids and Molluscs

Peptide

Sequence

Reference

Theromyzin

DHHHDHGHDDHEHEELTLEKIKEKIKDYADKTPVDQLTERVQAGRDYLLGKGARPSHLPARVDRHLSKLTAAEKQELADYLLTFLH

[165]

Chromacin

GDFELPSIADPQATFESQRGPSAQQVDK

[170]

T. tessulatum, along with the mussel, Mytilus edulis, was recently found to express invertebrate forms of peptide B [169]. Sequence comparisons of leech and mussel proenkephalin A showed that invertebrate forms of peptide B exhibited high levels of homology with those of vertebrates (Table 2). The peptide was detected in the haemocytes, neurons and haemolymph of T. tessulatum and M. edulis with these levels rapidly rising in response to surgical trauma and electrical shocks, which led to the suggestion that processing of the peptide in these invertebrates proceeds via intracellular and / or extracellular processes. Similar rises in levels of peptide B were observed when these organisms were injected with LPS, mimicking a bacterial challenge, and the peptide showed potent activity against M. luteus, with an MIC of circa 0.35 μM. The stimulated increases of peptide B detected in these experiments were accompanied by concomitant elevation in levels of the opioid Met-Enk (Fig. 1), which taken with other data, led to the suggestion that T. tessulatum and M. edulis possessed unified neuroimmune response mechanisms with many similarities to those found in vertebrates. Consistent with this suggestion, an anionic peptide (Table 6) showing circa 65% homology to the bovine chromacins described above (Table 1) was recently identified in T. tessulatum. The peptide exhibited a net charge of -3 and was rapidly released into the leech haemolymph in response to LPS-simulated bacterial challenge but in contrast to its bovine counterpart, showed no antibacterial activity. It was suggested that the peptide may play a signaling role in stimulation of the leech immune response [170]. Taken in combination, these observations led to the suggestion that vertebrates and invertebrates share a common immune response, and as invertebrates arose first during evolution, the molecules participating in this common immune response would have evolved from these primitive animals and then were retained over time [164]. Lysenins are a family of closely related anionic glycoproteins with a defence function that have been isolated from the coelomic fluid of the earthworm Eisenia fetida. Proteins of this family with known sequence include: lysenin / CL41 (Swiss-Prot accession code: O18423), lysenin-related protein 1 / hemolysin (Swiss-Prot accession code: O18424), lyseninrelated protein 2 / fetidin / CL39 / H1-3 (Swiss-Prot accession code: O18425) and lysenin-related protein 3 (Swiss-Prot accession code: Q3LX99), and a large number of isoforms of these AMPs are predicted to exist [171,172]. In general, these proteins are circa 300 residues in length and would appear to be amongst the largest AAMPs reported for eukaryotes. For convenience the sequences of these proteins have not been tabulated and can be found using the appropriate Swiss-Prot accession codes.

Fetidins have been shown to possess antibacterial activity and are found mixed with mucus as a covering on the outer body surface of E. fetida, thereby forming an antimicrobial barrier. A recombinant fetidin was found to inhibit the growth of B. megaterium, a bacterial strain reported to be pathogenic for E. fetida, and this ability was attributed to peroxidase activity based on the identification of a peroxidase signature within the primary structure of the protein [173]. The primary structure of lysenin was also found to possess a peroxidise signature and it was suggested that the protein may utilise a similar antibacterial mechanism [174]. However, more recent studies have shown that at nM levels, lysenin is active against B. megaterium and acts via membrane permeabilisation [171]. This result was surprising for numerous studies have shown that for efficient membrane insertion, lysenin requires the presence of sphingomyelin (SP), which acts as a high-affinity membrane receptor for the protein and is absent from bacterial membranes [175]. In response, it has been speculated that some component of the bacterial membrane such as ceramide or elements of the lipid A portion of LPS, may act a structural mimic of SP and thereby function as a receptor for lysenin [176]. However, the protein was found to show much slower kinetics for the lysis of bacterial membranes compared to SP-containing membranes and formed pores in the former membranes that were transient rather than the well-defined structures detected in the latter membranes [177,178]. Based on these observations, it was proposed that the toxicity of lysenin to bacterial membranes did not involve a specific receptor and that the protein had only low binding affinity for these membranes. Moreover, although clearly possessing antibacterial ability, lysenin showed no activity against soil bacteria, including the Gram-negative bacteria: P. aeruginosa, Serratia spp. or Sphingomonas spp, and at present, it is unclear as to whether antimicrobial activity is a primary defence role of the protein [171]. It has been suggested that targeting eukaryotic organisms may be the primary defence function of lysenin given that the presence of SP in membranes endows the protein with strong haemolytic ability along with cytotoxicity to vertebrate spermatozoa and amphibian larvae as well as cultured mammalian cells [179,180]. However, the defence function of lysenin cannot be fully explained in terms of the presence and absence of SP in membranes of the target organism as the protein is non-toxic to some invertebrates whose membranes contain the lipid [180]. Other factors are now known to affect the affinity of lysenin for SP [179] and further in depth discussion of the toxicity of lysenin to eukaryotes is beyond the scope of this review but it has been suggested that the protein might help defend E. fetida against predator


vertebrates or serve to protect discharged sperm of the earthworm, which lacks SP, from soil insects [174] It appears from the above studies that lysenin is able to serve a defence function in E. fetida by compromising the integrity of target cell membranes via SP-independent and SP-dependent mechanisms although many details of these mechanisms have yet to be elucidated. However, the SP– dependent interaction of lysenin is of great interest for it provides one of the few known examples of a specific lipid marker for proteins [175] and based on this observation, it has been suggested that the protein may form a new tool for investigating membrane lipid organization [181]. It has also been proposed that lysenin may serve as a probe for SP detection in SP storage diseases, particularly in cells of Niemann–Pick A patients, although the cytolytic activity of these proteins appears to be a major obstacle to this approach [174]. Whilst generally beyond the scope of this review, it is interesting to note that earthworms produce one of the relatively few examples of uncharged AMPs. Recent studies showed that E. fetida produced three peptides, F1, F2 and OEP3121, which comprised five hydrophobic residues, and exhibited potent activity against fungi, Gram-positive and Gram-negative bacteria with MICs in the low μM range [182,183]. These peptides were also of interest in that they would appear to be the shortest uncharged AMPs yet reported. Arachnids The Arachnids include spiders, mites, scorpions and ticks, which are parasites that such blood from vertebrate hosts for development and reproduction. In the process of feeding, ticks can become infected by a range of pathogenic micro-organisms and thereby transmit these pathogens. Despite their importance as major vectors in pathogen proliferation and disease transmission, the immune systems of these parasites are not well characterised [184]. In response, a differentially expressed cDNA library from synganglia of the tick, Amblyomma hebraeum, was used to investigate the genetic make-up of the arachnid and led to the identification of two putative AAMPs (Table 7). Sequence analyses showed that these peptides shared the cysteine motif of defensins from other ticks and on this basis, were named Amblyomma defensin peptide 1 (Swiss-Prot accession code: Q5VJF9), and Amblyomma defensin peptide 2 (Swiss-Prot accession code: Q5VJF8). The latter peptide possessed a net charge of -3 and when purified from the haemolymph of female ticks, was found to be active against Gram-negative and Grampositive bacteria with MICs in the low μM range. It was suggested that both Amblyomma defensin peptides may interact with the microbial membrane via cationic and hydrophobic loops at their C-termini. Screening for differentially expressed proteins revealed the upregulation of Amblyomma defensins during the 4 days after feeding by female ticks. This upregulation was accompanied by increasing levels of Amblyomma defensin 1 in the haemolymph of the organism but no corresponding increase in Amblyomma defensin 2 was detected. It was proposed that a regulatory pathway triggered by blood feeding may be involved in defensin gene expression by A. hebraeum and that identifica-

Harris et al.

tion of this pathway would help understanding of the immune mechanisms used by this tick [185]. More recently, cDNA encoding a putative AAMP (Table 7) was cloned and sequenced from the hard tick, Haemaphysalis longicornis. This protein was 59 residues in length with a net charge of -1 and possessed the cysteine motif associated with defensins. The peptide was found to be specifically expressed in the salivary gland of the organism and based on these observations was named Hlsal-defensin (Swiss-Prot accession code: A4GUC4). It was found that gene transcripts of Hlsaldefensin were significantly upregulated by the presence of LPS, which supported the view that the peptide was an inducible defence factor against bacterial infection. The antibacterial action of the Hlsal-defensin appeared to involve a 24 residue -sheet region of the AAMP, which when synthesised as a peptide homologue, was active against strains of S. aureus and E. coli with MICs in the low μM range [186]. These latter studies [185,186] will help provide understanding of the innate immunity of vector ticks and thus why some micro-organisms are transmitted by ticks and others not [184]. In studies on other members of the arachnid family, the use of cDNA libraries derived from the venom gland tissue of scorpions led to the description of a number of putative AAMPs. BmKa1 and BmKa2 (Table 7) were identified in Buthus martensii Karsch (Swiss-Prot accession codes: Q9Y0X5 and Q8N0N8), a Chinese scorpion whose venom has long been used in the traditional medicine of the country. The precursor proteins of these peptides were found to possess signal sequences characteristic of secreted molecules and mature BmKa1 and BmKa2 were found to be highly anionic with net charges of -12 and -13 respectively. These putative AAMPs exhibited no significant sequence homology with any other known proteins and were also unusual for scorpion venom peptides in that they contained no disulfide bridges. Secondary structure analysis predicted that BmKa1 was composed totally of random coil structures whilst BmKa2 contained three random coil regions separated by two -helical domains, which were potentially amphiphilic. No investigation into antimicrobial activity of these peptides was conducted but based on sequence similarities to other AAMPs, it was suggested that BmKa2 may represent a new class of defence peptides from scorpions [187]. Similar studies were conducted on the venom of the Brazilian scorpion, Tityus costatus Karsch, in response to the fact that this venom was previously uncharacterised and increasingly, humans were being stung by the creature. These studies identified four putative AAMPs (Table 7) named anionic peptide clones 7, 8, 9 and 10 (Swiss-Prot accession codes: Q5G8B0, Q5G8B1, Q5G8B2 and Q5G8A9), which were highly anionic with charges of -19, -19, -20 and -20, but no further characterization of these peptides appears to have been undertaken [188]. Insects One of the first cationic AMPs to be reported was in 1981 when cecropin was isolated from the haemolymph of pupae of the giant silk moth, Hyalophora cecropia. This led to the isolation of a large number of AMPs from moths and other insects [189,190] and development of the greater wax moth, Galleria mellonella, as a model organism for studies


Table 7.


AAMPs from Arachnids

Peptide

Sequence

Reference

Amblyomma defensin 1

FDNPFGCPADEGKCFDHCNNKAYDIGYCGGSYRATCVCYRK

[185]

Amblyomma defensin 2

YENPYGCPTDEGKCFDRCNDSEFEGGYCGGSYRATCVCYRT

[185]

DDSDHGFRTAHVDLVCPDNPDNCIQQCVSKGAQGGYCTNEKCTCYEKIPSATKRVRIVA

[186]

EENEEGSNESGKSTEAKNTDASVDNEDSDIDGDSD

[187]

Anionic peptide clone 10

PASYDGDFDALDDLDDLDLDDLLDLEPADLVLLDMWANMLDSQDFEDFE

[188]


PASYDDDFDALDDLDGLDLDDLLDSEPADLVLLDMWANMLDSQDFEDFE

[188]


PASYDDDFDALDDLDDLDLDDLLDLEPADLVLLDMWANMLDSQDFEDFE

[188]


PTSYDDDFDALDDLDDLDLDDLLDLEPADLVLLDMWANMLDSQDFEDFE

[188]

Hlsal-defensin BmKa1

Table 8.

597

AAMPs from Insects Peptide

Sequence

Reference

Gm anionic peptide 1

EADEPLWLYKGDNIERAPTTADHPILPSIIDDVKLDPNRRYA

[194]

Gm anionic peptide 2

ETESTPDYLKNIQQQLEEYTKNFNTQVQNAFDSDKIKSEVNNFIESLGKILNTEKKEAPK

[194]

VESWV

[146]

MDpep5

on innate immunity mechanisms [191,192]. Recent use of this model to characterise antimicrobial peptides in the haemolymph of immune-challenged G. mellonella larvae led to the description of two putative AAMPs (Table 8). These peptides were designated Gm anionic peptide 1 (Swiss-Prot accession code: P85211) and Gm anionic peptide 2 (SwissProt accession code: P85216). These AAMPs carried net charge of -4 and -3 respectively and showed activity against Gram-positive bacteria including: M. luteus and L. monocytogenes with MICs that were < 90 μM but were inactive against Gram-negative bacteria. In addition, each peptide showed narrow spectrum antifungal activity with MICs that were again < 90 μM. Sequence analysis showed that Gm anionic peptide 1 exhibited significant homology with segments from precursors of lebocins, which are weak antibacterial compounds from the silk moth, Bombyx mori [193], but as yet, it is not known if the AAMP is a G. mellonella lebocin-like peptide. In contrast, Gm anionic peptide 2 showed no homology to any other known protein and as yet, has not been further characterised [194]. Larvae of the housefly, Musca domestica, have long been used in Chinese medicine to treat bacterial infections and recently, extracts of these larvae have been shown to possess broad range antibacterial activity along with anticancer activity [195]. Attempts to characterise the antibacterial activity of these larvae led to the identification of cationic AMPs [196] and the shortest insect AAMP reported to date [197]. Designated MDpep5 (Table 8), this five residue peptide possessed a net charge of -1 and showed potent activity against a range of Gram-negative and Gram-positive bacteria with MICs that were < 55 μM. Studies on the antibacterial mechanism of MDpep5 found it able to bind to cells of E. coli and S. aureus and to partition into liposomes formed

from the endogenous lipid of these organisms. However, the presence of MDpep5 induced no significant leakage of cellular contents from E. coli and S. aureus cells and it was suggested that wholesale lysis of the cytoplasmic membrane by the peptide was unlikely to be the lethal event leading to bacterial death. The effect of the peptide on the surface potential of E. coli and S. aureus liposomes was then monitored and these data led to a proposed model for the antimicrobial action of MDpep5. According to this model, hydrophobicity drives the binding / partitioning of MDpep5 into bacterial membranes, which leads to increasing association of the peptide with the membrane and elevations in its negative surface potential. Eventually, this process leads to repulsive interactions between peptide molecules and at some critical concentration, MDpep5 forms aggregates with membrane lipid and translocates to the cell cytoplasm with cell resulting [197]. It has been suggested that MDpep5 may be of use in food preservation [197,198] given the current drive to reduce the utilisation of synthetic chemicals in this process and concerns over the microbial contamination of foodstuffs [199]. AAMPs FROM PLANTS The first AMP to be reported in a eukaryotic organism was wheat -purothionin, which was discovered in 1942, and over the last few decades, many more such peptides have been identified in various plant tissues, including: roots, leaves, seeds and flowers [4,200]. Based on these studies, it is a growing view that plants may share a common defence strategy with animals in that they synthesise and / or secrete AMPs in areas of initial contact with microbes. As with other eukaryotic organisms, the vast majority of these AMPs are cationic but increasingly, AAMPs are being reported in plant tissues [201]. For example, vicilins are seed storage proteins


Table 9.

Harris et al.

AAMPs from Plants Peptide

Sequence

Reference

MiAMP2a

ESEFDRQEYEECKRQCMQLETSGQMRRCVSQCDKRFEEDIDWSKYDNQD

[202]

MiAMP2b

DPQTDCQQCQRRCRQQESGPRQQQYCQRRCKEICEEEEEYN

[202]

EQCGRQAGGKLCPNNLCCSQWGWCGSTDEYCSPDHNCQSNCKD

[204]

WjAMP-1

QAGGQTCPGGICCSQWGYCGTTADYCSPNNNCQSNCWASG

[205]

Cn-AMP2

TESYFVFSVGM

[207]

Cn-AMP3

YCSYTMEA

[207]

Hevein

of the 7S globulin class and are present in the seeds of leguminous and other plants. These proteins are processed during the germination process, when plants are especially vulnerable to pathogenic interaction, and recently, several AAMPs were predicted from the processing of a 7S globulin protein in kernels of Macadamia integrifolia, the Macadamia nut. The first of these AAMPs (Table 9), MiAMP2a (Swiss-Prot accession code: Q9SPL3/4/5), was not characterized but interestingly, carried a net charge of -6 and so represents one of the most negatively charged plant AAMPs reported. The second of these putative AAMPs (Table 9), MiAMP2b (Swiss-Prot accession code: Q9SPL3/4/5), carried a net charge -2 and was isolated from seed exudate. The peptide was found to possess activity against fungal plant pathogens such as Leptosphaeria maculans and Verticillium dahliae with killing levels in the low μM range [202]. The antifungal mechanism of MiAMP2b was not investigated but it has recently been proposed that the toxic activity of vicilins is based on their ability to bind to the cell wall and the plasma membrane of fungi and yeasts [203]. These latter authors proposed a similar mechanism of action for hevein (Table 9) and hevein-like peptides, which is another established class of plant antifungal peptides. Studies on hevein (Swiss-Prot accession code: P02877), which was originally isolated from rubber latex of Hevea brasiliensis, the para rubber tree, showed that the peptide carried a net charge of -2 and was post-translationally cleaved from a precursor protein, prohevein. Investigation into the antimicrobial action of the peptide found that it inhibited the growth of chitin-containing fungi and associated strongly with chitin, which led to the suggestion that hevein may play a role in the protection of plant wound sites from fungal attack [204]. More recently, WjAMP-1 (Table 9), a hevein-like peptide with a net charge of -1 (Swiss-Prot accession code: Q8H950), was purified from leaves of Wasabia japonica. It was found that the inoculation of W. japonica with fungal pathogens induced expression of WjAMP-1 throughout tissues of the plant. The peptide showed activity against fungi and bacteria, and based on these observations, it was suggested that WjAMP-1 played a defence role in W. japonica [205]. Studies on leaves of bell pepper plants, Capsicum annuum, have suggested that anionic proteins and peptides are secreted onto plant leaf surfaces and may serve a role in the plant’s antimicrobial defence mechanisms. Extracts of these leaves were found to contain anionic peptides with potent antibacterial activity when directed against Raistonia so-

lanacearum and Clavibacter michiganensis spp, which are Gram-negative and Gram-positive bacterial plant pathogens respectively. A correlation between the levels of these AAMPs and antibacterial activity was observed with the highest level of activity found in cell wall extracts, consistent with a defence function in peripheral tissues vulnerable to pathogenic organisms [206]. More recently, AAMPs have been isolated from green coconut water, which is the clear liquid inside young coconuts and is a popular drink in the tropics, especially in Tropical Asia and Latin America. These peptides, designated Cn-AMP2 and Cn-AMP3 (Table 9), each exhibited a net charge of -1 and were found to be active against E. coli, B. subtilis, P. aeruginosa and S. aureus with MICs that were < 250 μM. It was predicted that Cn-AMP2 would adopt helical structure with a negatively charged surface region formed by the peptide’s sole charged residue, glutamic acid, and a hydrophobic patch due to the presence of residues such as phenylaniline. No clear structural predictions could be made for Cn-AMP3 but similarly to Cn-AMP2, the peptide possessed a negatively charged region due to the presence of its single glutamic acid residue and a non-polar surface, characterized by phenylalanine residues. It was suggested that the antimicrobial action of both peptides may involve insertion of their hydrophobic patches into the membrane of the target organism although the role of the negatively charged surface region was unclear [207]. One of the largest groups of AAMPs so far identified in plants are cyclotides (Table 10), which are distinguished by possession of a cyclic peptide backbone along with a knotted arrangement of three conserved disulphide bonds, generally termed a ‘cystine knot’ motif [208,209]. One of the first of these AAMPs to be characterized was kalata B2, which is derived from Oldenlandia affinis of the Rubiaceae [210,211], and was found to be active against the cotton pest, Helicoverpa armigera, suggesting an insecticidal role in plant defence [212,213]. More recently, the peptide has been shown to be active against the livestock gastrointestinal nematodes, Haemonchus contortus and Trichostronglyus colubriformis [214], and the Golden apple snail, Pomacea canaliculata [215]. Based on these studies, it was suggested that kalata B2 could be developed as a natural agent for pest control against nematode parasites and mollusks [214,215]. Since characterization of kalata B2, AAMPs of the cyclotide family have been increasingly reported in species of


the Rubiaceae, along with the Violaceae (Table 10), and have been shown to be haemolytic and highly effective against HIV [216-220]. Investigation into the insecticidal [213], antihelmintic [214], antiviral [216] and antimicrobial [221] action of cyclotides has suggested that the defence function of these peptides is underpinned by their ability to interact with membranes. Several structure / function studies have attempted to gain insight into this ability and have indicated that the membrane interactivity of cyclotides is primarily facilitated by hydrophobic areas on the surface of these peptides but that it is modulated by the relative location of their charged surface areas [216,217]. Some cyclotide, such as kalata B2, have a net negative charge and in these cases, surface areas formed by positively charged residues appear to help facilitate interaction of the peptide with the anionic moieties in the membranes of target microbes, which then leads to death of the organism [213]. However, some cyclotides only possess charged residues that are anionic and exhibit some of the highest levels of membrane interaction and antimicrobial activity reported. An example of these latter AAMPs is given by cycloviolacin Y4 (Table 10), which exhibits haemolytic and antiviral activities that are higher than those of the prototype cationic cyclotide, kalata B1. Currently, the role of the charged surface areas possessed by cycloviolacin Y4 and other such AAMPs in their membrane interactions and antimicrobial functions is unclear [216,217]. A number of therapeutic and biotechnical uses for cyclotides have been suggested: Cyclotide genes shows potential for incorporation as resistance factors into transgenic plants thereby protecting these plants from microbial pathogens, nematodes and insects. As well as improving the quality and production of food, transgenic plants hold great promise for the mass production of antibiotic and vaccines [209]. Cyclotides also show great potential for development as agents against HIV / AIDS, which remains one of the most important and preventable global causes of morbidity, mortality and disability, especially in the world's poorest countries [217]. In addition, cyclotides are showing great promise as a scaffold for the design of therapeutic drugs and protein engineering; the cystine knot framework of these peptides can accommodate a wide range of residue substitutions and they are noted for their stability to heat and chemical attack [222]. SUMMARY Since the first description of cationic AMPs in the early 1980s, there have been innumerable research papers, reviews and books devoted to these peptides. In contrast, AAMPs have received relatively little attention in the literature and in response we have presented an overview of current research into these peptides. This work has shown that upward of 100 AAMPs have been reported and it seems likely that this number will increase, particularly with the advent of genomic analysis. For example, recent sequencing of chromosome 2 of Arabidopsis thaliana, mouse-ear cress, has predicted the occurrence of thionin-2,3 (Swiss-Prot accession code: Q8VZK8), a 47 residue AAMP with a net charge of -2 [223]. Structural characterization shows AAMPs to generally range in net charge from -1 to -7 and in length from 5 residues to circa 70 residues although AAMPs with net charges of -20 [188] and length of 300 residues [171,172] have been


599

described. Some AAMPs are common to vertebrates and invertebrates, which shows the ancient nature of these peptides, and they have been identified in innate immune systems distributed across the eukaryotic kingdom indicating their importance to defence function. To perform their defence function AAMPs are generated by an array of mechanisms: Some AAMPs, such as human DCD, appear to be produced constitutively whilst others, such as leech theromyzin, appear to be induced in response to microbial infection. A number of inducible AAMPs, such as bovine enkelytin, are encrypted within the primary structures of precursor proteins and require cleavage by proteolytic cleavage. Interestingly, some peptides, such as TAPs from ruminants and humans, are cleaved from precursor proteins and function as AAMPs in a secondary capacity with other biological activities constituting their primary role. Presumably this dual function results from metabolical economy on the part of the host organism. The antimicrobial spectrum of AAMPs is broad with these peptides showing activity against bacteria, fungi, viruses and pests such as nematodes and insects. Based on the available experimental evidence, membrane interaction is expected to be a key step in the antimicrobial mechanisms of AAMPs. Some of these peptides, such as those derived from human DCD, have been speculated to interact with membranes via the use of receptors, but in general, these interactions appear to facilitated by the ability of AAMPs to adopt amphiphilic structures, including: -sheet, -helical and cyclic cystine knot architecture. For a number of AAMPs, post-translational modifications, such as phosphorylation, appear to be essential for their antimicrobial activity. These structural modifications have been speculated to stabilize the active conformation of bovine enkelytin and to facilitate the binding of divalent metal ions to bovine kappacins. These latter peptides, along with ruminant SAAPs and other AAMPS, appear to use metal ions to form cationic salt bridges with negatively charged components of microbial membranes, thereby facilitating interaction with their target organisms. However, the antimicrobial mechanisms of some AAMPs, such as amphibian maximin H5, have been shown to have no requirement for metal ions and in many cases, these mechanisms are unclear or have not yet been elucidated. Nonetheless, a wide array of uses for AAMPs has been suggested including: the diagnosis of sphingoyelin storage diseases, studies on interaction between the immune and nervous systems and as templates for drug design. However, the major use suggested for AAMPs are based on their antimicrobial activity and include: the treatment of ovine infections, disease control in fish farming, contraception and as topical biocides against sexually transmitted diseases, as food preservatives and as antibacterial additives to toothpaste and mouthwashes. Studies on scorpion toxins have suggested that AAMPs may be relics from the early evolution of antimicrobial peptides and that in the course of time the enhancement of toxicity to microbes has become associated with increases in the overall positive charge of antibiotics and toxins [224]. Other authors have suggested that AAMPs arose to complement cationic AMPs, providing a response to microbes that had developed resistance to these latter peptides for example [153]. Currently, the raison-d’être for AAMPs is a matter of debate but what is clear is that these peptides are an untapped source of antimicrobial agents with novel mechanisms of action, which is awaiting exploitation.


Harris et al.

Table 10. Cyclotide AAMPs from Plants Cyclotide

Sequence

Host plant

Swiss-Prot

Reference

Kalata B2

GLPVCGETCFGGTCNTPGCSCTWPICTRD

Oldenlandia affinis

P58454

[213]

Kalata B3

GLPTCGETCFGGTCNTPGCTCDPWPICTD

Oldenlandia affinis

P58455

[225]

Kalata B4

GLPVCGETCVGGTCNTPGCTCSWPVCTRD

Oldenlandia affinis

P83938

[225]

Kalata B10

GLPTCGETCFGGTCNTPGCSCSSWPICTRD

Oldenlandia affinis

P85128

[225]

Kalata B11

GLPVCGETCFGGTCNTPGCSCTDPICTRD

Oldenlandia affinis

P85129

[225]

Kalata B12

GSLCGDTCFVLGCNDSSCSCNYPICVKD

Oldenlandia affinis

P85130

[225]

Kalata B13

GLPVCGETCFGGTCNTPGCACDPWPVCTRD

Oldenlandia affinis

P85131

[225]

Kalata B14

GLPVCGESCFGGTCNTPGCACDPWPVCTRD

Oldenlandia affinis

P85132

[225]

Kalata B15

GLPVCGESCFGGSCYTPGCSCTWPICTRD

Oldenlandia affinis

P85133

[225]

Kalata B16

GIPCAESCVYIPCTITALLGCKCQDKVCYD

Oldenlandia affinis

P85134

[225]

Cyclovialacin-O23

GLPTCGETCFGGTCNTPGCTCDSSWPICTHN

Viola odorata

P85186

[218]

Cyclovialacin-O24

GLPTCGETCFGGTCNTPGCTCDPWPVCTHN

Viola odorata

P85187

[218]

Cycloviolacin-H3

GLPVCGETCFGGTCNTPGCICDPWPVCTRN

Viola hederaceae

P85232

[220]

Cycloviolacin-H4

GIPCAESCVWIPCTVTALLGCSCSNNVCYN

Viola hederaceae

P85234

[219]

Vh1-2

GLPVCGETCFTGTCYTNGCTCDPWPVCTRN

Viola hederaceae

P85231

[220]

Hyfl-B

GSPIQCAETCFIGKCYTEELGCTCTAFLCMKN

Hyabanthus floribundus

P84648

[226]

Vary peptide B

GLPVCGETCFGGTCNTPGCSCDPWPMCSRN

Viola arvensis

P58447

[227]

Vary peptide G

GVPVCGETCFGGTCNTPGCSCDPWPVCSRN

Viola arvensis

P58452

[227]

Vary peptide H

GLPVCGETCFGGTCNTPGCSCETWPVCSRN

Viola arvensis

P58453

[227]

Palicourein

GDPTFCGETCRVIPVCTYSAALGCTCDDRSDGLCKRN

Palicourea condensata

P84645

[228]

Cycloviolacin Y4

GVPCGESCVFIPCITGVIGCSCSSNVCYLN

Viola yedoensis

[216]

Cycloviolacin Y5

GIPCAESCVWIPCTVTALVGCSCSDKVCYN

Viola yedoensis

[216]

ACKNOWLEDGEMENTS Dr Frederick Harris would like to acknowledge the support given by Dr. Lynne Pierpoint who has provided him with true inspiration over recent months.

MBCs

= Minimum bactericidal concentrations

MIC

= Minimum inhibitory concentration

NEP

= Neutral endopeptidase

ABBREVIATIONS

PCs

= Proprotein convertases

PEA

= Proenkephalin A

ACE

= Angiotensin converting enzyme

AD

= Atopic dermatitis

AMPs

= Antimicrobial peptides

AAMPs = Anionic antimicrobial peptides

SAAPS = Surfactant-associated anionic peptides SP

= Sphingomyelin

TAPs

= Trypsinogen activation peptides.

CGA

= Chromogranin A

REFERENCES

CNS

= Central nervous system

[1]

DCD

= Dermcidin

[2]

HIV

= Human immunodeficiency virus

HSV

= Herpes simplex virus

LPS

= Lipopolysaccharide

[3]

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Received: May 21, 2009

Revised: July 02, 2009

Accepted: July 06, 2009

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