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The expanding scope of antimicrobial peptide structures and their modes of action
Review
The expanding scope of antimicrobial peptide structures and their modes of action Leonard T. Nguyen, Evan F. Haney and Hans J. Vogel Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada, T2N 1N4
Antimicrobial peptides (AMPs) are an integral part of the innate immune system that protect a host from invading pathogenic bacteria. To help overcome the problem of antimicrobial resistance, cationic AMPs are currently being considered as potential alternatives for antibiotics. Although extremely variable in length, amino acid composition and secondary structure, all peptides can adopt a distinct membrane-bound amphipathic conformation. Recent studies demonstrate that they achieve their antimicrobial activity by disrupting various key cellular processes. Some peptides can even use multiple mechanisms. Moreover, several intact proteins or protein fragments are now being shown to have inherent antimicrobial activity. A better understanding of the structure– activity relationships of AMPs is required to facilitate the rational design of novel antimicrobial agents. Introduction The emergence of multidrug-resistant ‘superbug’ bacteria has created an urgent need for the development of novel classes of antimicrobials. Unfortunately, the number of new antibiotics in the pipeline of the big pharmaceutical companies has been declining because they have shifted their attention towards more lucrative areas of drug development [1]. Since the initial discovery of antimicrobial peptides (AMPs) in insects and animals in the 1980s, they have been heralded as a promising alternative to today’s antibiotics [2]. Ubiquitous in nature, AMPs form a key component of an organism’s innate immune system. AMPs typically have a broad spectrum of activity against pathogenic bacteria and fungi, and often, their killing activities extend to enveloped viruses, parasites and sometimes even to cancerous cells. Many eukaryotic peptides act on bacterial membranes or other generalized targets, in contrast to most antibiotics, which usually target specific proteins. This creates an advantage for AMPs because development of microbial resistance by gene mutation is less likely [3]. AMPs obtained from higher eukaryote organisms are also frequently referred to as ‘host defense peptides’, a term that emphasizes their additional immunomodulatory activities [2]. Traditionally, the search for novel AMPs involved the identification of active peptides from natural sources. Such studies are followed by the design of synthetic peptide Corresponding author: Vogel, H.J. ([email protected]).
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analogs for structure–function studies. De novo peptide design approaches have also been used, including highthroughput combinatorial library screening, structurebased modeling, predictive algorithms and the introduction of non-coded modifications to conventional peptide chemistry [4,5]. Furthermore, AMPs are being discovered through sequence searches for potential antimicrobial fragments within large proteins. In fact, several proteins have now been shown to exert direct antimicrobial activity themselves, further widening the scope of the structural frameworks for AMPs or antimicrobial proteins. Generally, two physical features are common for AMPs: a cationic charge and a significant proportion of hydrophobic residues. The former property promotes selectivity for negatively charged microbial cytoplasmic membranes over zwitterionic mammalian membranes whereas the latter facilitates interactions with the fatty acyl chains [6]. There are also a few anionic AMPs, such as dermcidin, although other biological activities seem to be more important for these peptides [7]. Many linear AMPs are unstructured in aqueous solution and require a membranous environment to adopt a stable amphipathic conformation [8]. Although the mode of action usually involves disrupting the integrity of the bacterial cytoplasmic membrane in many ways (Figure 1), other antimicrobial mechanisms have now been characterized that target key cellular processes including DNA and protein synthesis, protein folding, enzymatic activity and cell wall synthesis [9–11]. A comprehensive list of mechanisms utilized by AMPs is presented in Table 1. Membrane interactions remain important even for intracellular-targeting peptides because they must have a means of translocation. The cationicity of the AMPs also promotes interactions with negatively charged moieties on other biomolecules such as outer membrane lipids, nucleic acids and phosphorylated proteins. During an infection, a host animal may release multiple isoforms or structurally similar AMPs that act by distinct mechanisms to achieve an overall synergistic effect [12]. Furthermore, for some of the better studied AMPs, such as magainin and indolicidin, it has been found that they can have multiple modes of action, suggesting that they may work as ‘dirty’ antimicrobials that rely on a multiple-hit strategy to increase their efficiency and evade potential resistance mechanisms [3]. In this review, we aim to illustrate the wide diversity of structural properties demonstrated by AMPs as well as the growing number of ways in which they exert their activities.
0167-7799/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2011.05.001 Trends in Biotechnology, September 2011, Vol. 29, No. 9
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Toroidal pore Disordered toroidal pore Carpet model +
Membrane adsorption
Barrel stave +
+
+
+
+
Conformational change
Δd
Membrane thinning/thickening
+
0.2V
? Electroporation Charged lipid clustering Bacterial membrane ΔΨ
O Non-lytic membrane depolarization
+
+
H
+
-
Non-bilayer intermediate
Anion carrier
Oxidized lipid targeting TRENDS in Biotechnology
Figure 1. Events occurring at the bacterial cytoplasmic membrane following initial antimicrobial peptide (AMP) adsorption. These events are not necessarily exclusive of each other. In the classical models of membrane disruption, the peptides lying on the membrane reach a threshold concentration and insert themselves across the membrane to form either peptide-lined pores in the barrel-stave model, solubilize the membrane into micellar structures in the carpet model, or form peptide-and-lipidlined pores in the toroidal pore model. In the revised disordered toroidal pore model, pore formation is more stochastic and involves fewer peptides. The thickness of the bilayer can be affected by the presence of the peptides, or the membrane itself can be remodeled to form domains rich in anionic lipids surrounding the peptides. In more specific cases, non-bilayer intermediates in the membrane can be induced; peptide adsorption to the membrane can be enhanced by targeting them to oxidized phospholipids; a peptide may couple with small anions across the bilayer, resulting in their efflux; the membrane potential can be dissipated without other noticeable damage; or conversely, in the molecular electroporation model, the accumulation of peptide on the outer leaflet increases the membrane potential above a threshold that renders the membrane transiently permeable to various molecules including the peptides themselves.
Recent structure–function relationship studies will be presented within the framework of the three AMP structural groups: a-helical peptides, b-sheet peptides and extended peptides (Figure 2) [13]. a-Helical AMPs The a-helical AMPs, such as magainin from the African clawed frog, are amongst the most intensely studied AMPs [14,15]. Numerous structure–function studies have indicated that membrane-bound a-helices with too large a hydrophobic surface become cytotoxic to mammalian cells [16]. Although these peptides are characterized by their structure, an extremely high propensity for the formation of the a-helix, which may present a more continuous hydrophobic surface, can become a contributing factor to cytotoxicity [17]. Indeed, peptides that remain antimicrobial but have low cytotoxicity require a membranous environment to induce proper folding and often have a proline- or glycine-induced
kink in the middle of the a-helix. The amphipathicity of the peptides is usually segregated along the axis of the a-helix such that the peptide can lie parallel to the membrane plane during the initial lipid interactions, with the charged side facing outward towards the phospholipid head groups and the hydrophobic side embedded into the acyl tail core. The properties of the a-helix tails sometimes influence the depth of membrane insertion and, in turn, the antimicrobial activity [18]. The lengths of the a-helices are usually sufficient to span the thickness of the membrane bilayers, with mismatches between the hydrophobic portion of the helices and the lipid acyl core strongly influencing the AMPs’ lateral and orientational motions in the membranes [19,20]. The peptides are thought to disrupt bacterial membranes through the formation of toroidal pores composed of loosely associated peptides with interdigitating phospholipid head groups among them [10]. Pore formation is often associated with a local change in 465
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Table 1. Different modes of action that have been described for AMPs Major target External proteins Outer surface lipids
Outer membrane proteins (Gram-negative)
Inner membrane
Integral membrane proteins Nucleic acids
Intracellular proteins
Specific target/mode of action Autolysin activation Phospholipase A2 activation LPS permeabilization (Gram-negative) Lipid II (peptidoglycan precursor)
Example peptides a Pep5, nisin Magainin 2, indolicidin Cecropin P1 Defensins, Nisin and other lantibiotics
Outer membrane protein I (OprI) LPS-assembly protein D (LptD) inhibition Outer membrane protein F (OmpF) Barrel-stave pore Detergent micellization Toroidal pore Disordered toroidal pore Membrane thinning/thickening Charged lipid clustering Non-bilayer intermediate formation Oxidized phospholipid targeting Anion carrier Non-lytic membrane depolarization
SMAP-29, CAP-18 Protegrin I peptidomimetic analogs HP(2–20) analog Alamethicin Dermaseptin, cecropin Magainin 2, melittin protegrin I Magainin analog, melittin PGLa, LL-37 Magainin analogs, Arg-rich peptides Gramicidin S Temporin L, indolicidin Indolicidin Bovine lactoferricin, daptomycin
Electroporation Proton translocation-related proteins DNA (general) DNA (covalent interaction) Branched DNA RNA (general) DnaK inhibition, GroEL chaperonin 20S proteasome, SH3-containing proteins
NK-lysin Clavanin A Buforin 2, tachyplesin, indolicidin Indolicidin WRWYCR Buforin 2 Pyrrhocoricin, drosocin, apidaecin PR-39
Refs [10] [10] [69] [34,35] [70] [71] [33] [72] [10] [10] [10] [31] [22] [21] [26] [73] [27] [49] [32] [74] [42] [28] [10] [50] [43] [10] [10] [46] [47]
a
Peptides listed are not limited to their associated modes of action here and may employ multiple mechanisms simultaneously.
membrane thickness [21]. Recent molecular dynamics (MD) simulations with a magainin analog and with melittin have suggested that a re-evaluation of this model is in order [22,23]. In the ‘disordered toroidal pore’, lipid molecules are still curved inwards, but pore formation is more stochastic and only one or two peptides are located near the center of the water-permeable pore, with more peptides positioned at the mouth of the pore on the external leaflet. As they translocate to the interior of the cell, the peptides remain mostly parallel to the bilayer plane, and only a small tilt is observed. Interestingly, the peptides undergo partial unfolding during translocation and their a-helicity is not always maintained. In other MD simulations, similar tilted orientations for a-helical peptides were observed and the notion of imperfect amphipathicity was introduced [24]. This refers to the presence of some polar or charged residues in the hydrophobic face of an AMP to pull lipid head groups into the membrane interior, thereby facilitating pore formation. A perfect hydrophobic face on a peptide would favor a fixed parallel orientation on a flat membrane surface. Imperfect amphipathicity is also a highlighted feature in the proposed interfacial activity model [25], which is based on a recent review of AMP literature. It closely resembles the disordered toroidal pore model, with the key difference of the interfacial activity model describing membrane disruption without a need for peptide self-assembly. The carpet model, where AMPs act more in a detergentlike manner to break off the membrane lipids into micellelike structures, has also been described. This model 466
appears to be applicable mostly to the high concentrations used in vitro, and is generally viewed as an extreme extension of the toroidal pore model [10]. An interesting membrane perturbing effect induced by some a-helical AMPs is that of lipid segregation, where peptide-induced clustering of anionic lipids in bacterial membranes causes slow leakage of intracellular contents and/or membrane depolarization [26]. Alternatively, the phase boundary effects between domains of differently charged lipids may compromise the overall membrane stability. Moreover, many bacterial membranes are mostly composed of phosphatidylglycerol and phosphatidylethanolamine lipids, where the latter does not like to form stable bilayers after peptide-induced demixing. In the case of Gram-positive bacteria such as Staphylococcus aureus, which have cytoplasmic membranes that are mainly composed of the negatively charged lipids phosphatidylglycerol and cardiolipin, lipid segregation is not expected and this is reflected in lower toxicity of some AMPs against these species [26]. The targeting of oxidized lipids represents a unique membrane-associated means for AMPs to enhance their activity. The a-helical AMPs temporin B and L intercalate more efficiently into membranes containing an oxidized phosphatidylcholine lipid, probably via Schiff base formation between the peptide amino groups and lipid aldehyde groups [27]. With the release of reactive oxygen species during phagocytosis, it is possible that lipid oxidation could increase bacterial membrane susceptibility to AMPs.
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(a)
LL-37
Bovine lactoferrampin
Magainin 2
(b)
Bovine lactoferricin
Protegrin I Human β-defensin-3
(c)
RRWQWR Tritrpticin
Indolicidin TRENDS in Biotechnology
Figure 2. An overview of the major structural classes of antimicrobial peptides (AMPs). (a) a-Helical peptides, (b) b-sheet peptides and (c) extended peptides. All of these structures were solved by solution NMR spectroscopy in the presence of detergent micelles, except for the b-sheet peptides, which were studied in aqueous solution. Positively charged side chains are colored in blue, negatively charged side chains in red and remaining side chains in grey. PDB IDs: magainin 2, 2MAG; LL-37, 2K6O; bovine lactoferricin, 1LFC; protegrin 1, 1PG1; human b-defensin-3, 1KJ5; tritrpticin, 1D6X; indolicidin, 1G89.
Finally, the Phe-, His- and Gly-rich peptide clavanin A has a unique dual pH-dependent mode of action [28]. At neutral pH, it permeabilizes membranes like regular ahelical AMPs. At slightly acidic pH, clavanin A is still membrane disruptive. However, this does not occur via lipid interactions, but rather through inhibitory interactions with proteins involved in maintaining the pH gradient across the membrane. In contrast to most a-helical peptides, buforin II, a peptide derivative from histones of the Asian toad Bufo bufo gargarizans, does not cause membrane disruption [9] although its cell entry can be affected by membrane lipid composition [29]. After translocation, buforin II accumulates in the cytoplasm where it is known to exert its antimicrobial activity by adapting an extended or polyproline II conformation to accommodate interactions with nucleic acids [30]. b-Sheet AMPs The b-sheet AMPs include several b-hairpin peptides in addition to the defensin mini-proteins. Many of these peptides disrupt bacterial membranes via the formation of toroidal pores as observed for the porcine peptide protegrin I. This peptide forms oligomeric transmembrane bbarrels in anionic membranes, but forms b-sheet aggregates on the surface of cholesterol-containing membranes [31].
Additional evidence suggests that non-lytic mechanisms are also employed by certain b-sheet peptides. Although tachyplesin from horseshoe crabs is generally recognized as a membrane active peptide, it is also capable of binding to the minor groove of DNA, which may interfere with DNA–protein interactions [10]. The b-hairpin peptide bovine lactoferricin can act synergistically with other antimicrobial agents by affecting the transmembrane potential and proton-motive force, resulting in inhibition of ATPdependent multi-drug efflux pumps [32]. Following translocation, bovine lactoferricin localizes to the cytoplasm where it can also inhibit DNA, RNA and protein synthesis. A series of peptidomimetic analogs of protegrin I featuring stabilized b-hairpins are no longer membrane active, but employ a new bactericidal mechanism involving a specific protein target that affects outer membrane biosynthesis [33]. Although these peptide analogs are remarkably potent, their spectrum of activity is narrowed to Pseudomonas aeruginosa strains, which express the susceptible homolog of this b-barrel protein. Finally, although the human defensins are membrane active, some of them can also bind to the peptidoglycan precursor lipid II to inhibit cell wall biosynthesis enzymes in Staphylococci [34,35]. The b-hairpin or b-sheet conformations of these peptides are often stabilized by disulfide bridges between conserved Cys residues. However, these covalent bonds 467
Review do not appear necessary for antimicrobial activity. Linear derivatives of tachyplesin and lactoferricin retain antimicrobial activity while losing hemolytic activity [32,36]. Solid state NMR studies of tachyplesin and two linearized analogs, in which the four Cys residues were substituted by either Phe or Ala residues, show that all three peptides are positioned at the membrane interface and adopt similar bsheet conformations on anionic membranes [37]. However, this does not correlate with their activities: the Cys-to-Phe peptide is equally active as tachyplesin whereas the Cysto-Ala peptide is inactive. Peptide dynamics, rather than their structures, provide a reasonable explanation for these functional differences. Whereas the active tachyplesin peptide shows enough mobility in the liquid-crystalline phase of the membrane to disturb lipid packing, the inactive analog is immobilized on the membrane and stays highly aggregated. At 45 residues, human b-defensin-3 (HBD-3) is rather large as an AMP, containing an a-helical turn followed by a three-stranded antiparallel b-sheet. This scaffold is held together by three disulfide bonds in a defined pattern that distinguishes the b-defensins from the a-defensins. Nonetheless, rearranging the disulfide pattern in HBD-3 or reducing them does not alter the antimicrobial activity, nor does it abolish the overall formation of the native secondary structure elements [38,39]. Although antimicrobial activity may be unaffected, chemotactic activities are decreased for the analogs with non-native disulfide linkages. Interestingly, reducing the disulfides in HBD-1 creates a flexible unstructured peptide with improved activities against Candida albicans and some Gram-positive bacteria [40]. The presence of reduced HBD-1 could be of great physiological relevance in the anaerobic environment of the colon. Extended AMPs Extended AMPs, which do not fold into regular secondary structure elements, often contain high proportions of certain amino acids, specifically Arg, Trp or Pro residues. The His-rich salivary histatin peptides are sometimes considered to be extended AMPs, however they are not bacteriostatic nor bactericidal despite being fungicidal and antiparasitic [41]. Many of the extended peptides are not membrane active. The Pro-rich insect-derived pyrrhocoricin, drosocin and apidaecin peptides penetrate across the membrane and exert their antimicrobial activities by making interactions with intracellular proteins such as the heat-shock protein DnaK and GroEL to inhibit the DnaK ATPase activity and chaperone-assisted protein folding, respectively [10]. Trp and Arg side chains are prominent in peptides with less than 15 residues [42]. Two of the shortest active peptides include RRWQWR and RAWVAWR, which have been identified as the active antimicrobial fragments from bovine lactoferricin and human lysozyme, respectively. In addition, combinatorial peptide library screening has identified the hexameric sequence RRWWRF as having potent broad spectrum activity [4]. Although these peptides can fold into defined amphipathic molecules in the presence of a membrane, they are not membrane active and, instead, 468
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they accumulate in the cytoplasm. The hexapeptide WRWYCR, which was identified as a DNA-repair inhibitor that binds to Holliday junctions, was subsequently shown to have broad bactericidal activity [43]. Given the sequence similarity of this peptide to those mentioned above, their efficacy may be due to this mechanism, where trapping of the replication fork prevents recombination and DNA repair. The 13-residue Arg- and Trp-rich tritrpticin and indolicidin peptides from porcine and bovine leukocytes, respectively, can cause membrane leakage [42]. In a membranous environment, the structures of both peptides feature multiple Pro-induced turns that give them amphipathic boat-like conformations. These peptides are sufficiently small that simple residue substitutions can have large consequences for their structural and functional properties. Replacing both Pro residues with Ala in tritrpticin transforms it into an a-helical peptide with slightly improved membrane leakage and antimicrobial activity, but also higher cytotoxicity [44]. The role of the two Pro residues is distinct, with Pro5 having more of a structural role preventing the induction of a-helical structure. Ironically, the characteristic Arg and Trp side chains do not seem an optimal choice of residues for tritrpticin. In analogs with Arg-to-Lys and Trp-to-Phe substitutions, the antimicrobial activity is maintained and the slight hemolytic activity of tritrpticin is abolished. The reduced hydrogen bonding capabilities of Lys compared to the Arg guanidinium group seem to increase membrane selectivity for the peptide. Replacement of the three Trp residues with Phe created a tritrpticin variant that was no longer able to adopt a single well-defined conformation in a membrane [44]. Interestingly, this peptide required a few hours to take effect whereas native tritrpticin completes bacterial killing in less than one hour [45]. The fact that multiple mechanisms are in play is further substantiated by the synergistic effects observed between the Trp-to-Phe and membrane active Pro-to-Ala peptides [45]. The Pro- and Arg-rich porcine PR-39 peptide interacts with cytoplasmic proteins such as the 20S proteasome and SH3-domain proteins, and it is believed that this contributes to its antimicrobial mode of action [46,47]. Indolicidin has been a template for many analogs with improved biological properties, the most successful one being omiganan, which was developed by Migenix (renamed to Biowest Therapeutics) and has entered phase III clinical trials [48]. A number of contributing mechanisms may explain the efficiency of indolicidin. This includes the activation of phospholipase A2 secreted by human lacrimal fluid to hydrolyze anionic membranes [10] or a direct membrane action either through lipid segregation, the targeting of oxidized phospholipids or acting as a small anion carrier across the membrane [27,49]. Moreover, at the intracellular level, both covalent and noncovalent DNA–indolicidin interactions can interfere with DNA-binding enzymes and induce filamentation in Escherichia coli [50]. It is unclear how the short and unique sequence of indolicidin, which includes five Trp residues and three Pro-induced turns, can exhibit all these activities and which of these proposed mechanisms contributes most to its activity in vivo.
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Protein-derived antimicrobial fragments and antimicrobial proteins AMPs can be derived from large proteins. In addition, several cationic proteins have been discovered to possess direct nonenzymatic antimicrobial activity themselves, some of which are shown in Figure 3. The primary function of these antimicrobial proteins is usually immunity-related, therefore they show significant functional overlap with AMPs (Table 2). Lactoferrin (Lfin) is a multifunctional 80 kDa glycoprotein present in biofluids, most notably in milk and neutrophil granules. The protein is involved in several immunoregulatory processes that primarily take advantage of its strong antimicrobial properties. This activity is due in part to its ability to sequester iron, which prevents bacterial growth. However, Lfin also has an iron-independent mechanism arising from direct interactions with bacterial cell components [51]. Three cationic antimicrobial fragments have been identified in different parts of the Lfin sequence: lactoferricin, which is a naturally occurring pepsin digestion product, lactoferrampin and kaliocin (Figure 3a) [18,32,52]. The structural differences for the isolated peptides and the corresponding regions in Lfin are significant; in particular, bovine lactoferricin and kaliocin form amphipathic b-hairpin peptides, whereas lactoferrampin requires a membraneous environment to adopt its native a-helical structure. The intact protein and the peptides probably kill bacteria in a different manner, especially lactoferricin, which is substantially more active than intact Lfin.
[(Figure_3)TD$IG]
Whether they are naturally released proteolysis products or designed synthetic peptides, many AMPs have been derived from proteins such as casein [53], lysozyme and ovotransferrin [54], myoglobin and hemoglobin [55], histones [9], growth factors [56], and thrombin [57]. Thus, scanning the proteome for related sequences with appropriate combinations of cationic and hydrophobic residues may uncover additional AMPs. Some of these proteins such as lysozyme and histones already have intrinsic antimicrobial activity and, as is the case for Lfin, their peptide fragments are expected to have different modes of action [58]. Recently, more than 20 human chemokine proteins have been shown to be directly antimicrobial, with macrophageinflammatory protein-3a (MIP-3a) possessing the most potent and broadest spectrum of activity (Figure 3b) [59]. In the case of the chemokine CCL28, the antimicrobial mechanism has been identified as membrane permeation [60]. Conversely, a few defensins are now known to possess chemotactic activity [61]. A common and unifying feature of the antimicrobial chemokines is the presence of a large uninterrupted cationic surface. Some chemokines tend to dimerize under physiological conditions, further extending the cationic surface [62]. For example, HBD-3 has a stronger tendency to dimerize, which may contribute to its high salt-insensitive activity compared to other b-defensins [63]. The C-terminal a-helices of several antimicrobial chemokines have multiple positively charged residues and when synthetic peptides of this region are studied, they behave like membrane-active a-helical AMPs [64].
(a)
(b)
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Figure 3. The structures of immunity-related proteins with direct antimicrobial activity and antimicrobial fragments. (a) Human lactoferrin (PDB ID 1FCK) with segments corresponding to its antimicrobial fragments: lactoferricin in blue, kaliocin in red and lactoferrampin in yellow (left panel). The electrostatic potential surface plot of lactoferrin is shown in the right panel. (b) Macrophage inflammatory protein-3a (PDB ID 2JYO) with the MIP-3a51–70 peptide segment colored in blue (left panel). The electrostatic potential surface plot of the monomeric unit of MIP-3a is shown in the middle panel and the same plot for the MIP-3a dimer is in the right panel, showing the stronger cationic surface around the self-associating a-helices.
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Table 2. A selection of human AMPs and antimicrobial proteins highlighting their multifunctionality in the immune system Main sources
Indirect antimicrobial and additional functions a
Refs
AMPs LL-37
Leukocytes, epithelia
[61]
Defensins
Leukocytes, epithelia
Dermcidin Histatins Lactoferricin
Skin Saliva Digested milk protein
Endotoxin neutralization, chemotaxis, reactive oxygen species (ROS) formation, wound healing, mast cell degranulation Endotoxin neutralization, opsonization, chemotaxis, ROS formation, wound healing, complement inhibition Chemotaxis Wound healing Endotoxin neutralization, complement inhibition, transcription factor
Antimicrobial proteins Chemokines Neutrophil serprocidins (azurocidin, cathepsin G, elastase) Bactericidal/permeability increasing protein Lipocalins Lysozyme Lactoferrin
Neutrophils, secretory fluids Secretory fluids, neutrophils Secretory fluids, neutrophils
RNAse 7 Psoriasin Histones Prion protein
Skin Skin Nuclei Brain, other tissues
Leukocytes Neutrophils Neutrophils
Chemotaxis Protease, ROS formation, opsonization, macrophage activation Endotoxin neutralization Siderophore sequestering Peptidoglycan digestion Iron sequestering, endotoxin neutralization, phagocytosis stimulation Ribonuclease Neutrophil activation, ROS formation Chromatin regulation Unknown
[61] [7] [75] [32]
[59] [61] [61] [76] [61] [61] [77] [77] [78] [79]
a
These are usually secondary functions for AMPs and primary functions for antimicrobial proteins.
This property may explain the bactericidal properties of several intact chemokines; however, some of the a-helical peptides do not retain activity and, furthermore, some chemokines do not have cationic C-terminal regions despite displaying activity. For the platelet chemokine neutrophil activating protein-2 (NAP-2), the proteolytic removal of the two C-terminal Ala-Asp residues leads to a drastic increase in the antimicrobial potency. This was attributed to the negatively charged Asp side chain interfering with a positive patch on the protein surface that includes side chains from the C-terminal a-helix as well as the b-sheet loops [65]. Concluding remarks Widely different structural scaffolds seem to support the activity of AMPs and proteins, with amphipathicity remaining as the only overarching physical property. A combination of a well-defined cationic region together with an imperfect hydrophobic surface area frequently seems to result in broad spectrum activity. In addition, the dynamic properties of these peptides can affect their biological properties, with structural rigidity generally hindering activity. Initially, AMPs were considered as a uniform group of molecules that ‘punch holes’ in bacterial membranes. In recent years, it has become apparent that their mechanism of action is much more complex and diverse. Current research efforts now also include antimicrobial fragments from intact proteins and proteins with direct antimicrobial activity, forcing structural biology researchers to reevaluate the common properties within this expanding family. Clearly, host defense systems have developed a broad arsenal and use a multi-pronged strategy to combat invading pathogens. Many AMPs seem to be capable of killing bacterial cells in multiple ways, making it a difficult task to unravel all the molecular events that occur when a peptide encounters a 470
bacterial cell. Consequently, the design and prediction of peptide analog characteristics is not straightforward and all peptides and analogs need to be studied on a case-by-case basis. Apart from their biological activities, other aspects need to be considered when developing these peptides for systemic use, including in vivo stability [66], side effects and production costs. Based on our current understanding of the structure–function relationships for different classes of AMPs, some non-peptide and peptidomimetic compounds have been developed and have advantageous properties because they combine stability with high antimicrobial potency [67,68]. Overall, detailed structure–function and biophysical studies can rationalize why a certain peptide is more active or behaves differently from a related peptide or an analog. Such comprehensive understanding will be essential to guide the design of better AMPs. Acknowledgments Research on AMPs in the H.J. Vogel laboratory is supported by a grant from the Novel Alternatives for Antibiotics Program of the Canadian Institute for Health Research. H.J.V. is the holder of a Scientist award from the Alberta Heritage Foundation for Medical Research.
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The expanding scope of antimicrobial peptide structures and their modes of action Leonard T. Nguyen, Evan F. Haney and Hans J. Vogel Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada, T2N 1N4
Antimicrobial peptides (AMPs) are an integral part of the innate immune system that protect a host from invading pathogenic bacteria. To help overcome the problem of antimicrobial resistance, cationic AMPs are currently being considered as potential alternatives for antibiotics. Although extremely variable in length, amino acid composition and secondary structure, all peptides can adopt a distinct membrane-bound amphipathic conformation. Recent studies demonstrate that they achieve their antimicrobial activity by disrupting various key cellular processes. Some peptides can even use multiple mechanisms. Moreover, several intact proteins or protein fragments are now being shown to have inherent antimicrobial activity. A better understanding of the structure– activity relationships of AMPs is required to facilitate the rational design of novel antimicrobial agents. Introduction The emergence of multidrug-resistant ‘superbug’ bacteria has created an urgent need for the development of novel classes of antimicrobials. Unfortunately, the number of new antibiotics in the pipeline of the big pharmaceutical companies has been declining because they have shifted their attention towards more lucrative areas of drug development [1]. Since the initial discovery of antimicrobial peptides (AMPs) in insects and animals in the 1980s, they have been heralded as a promising alternative to today’s antibiotics [2]. Ubiquitous in nature, AMPs form a key component of an organism’s innate immune system. AMPs typically have a broad spectrum of activity against pathogenic bacteria and fungi, and often, their killing activities extend to enveloped viruses, parasites and sometimes even to cancerous cells. Many eukaryotic peptides act on bacterial membranes or other generalized targets, in contrast to most antibiotics, which usually target specific proteins. This creates an advantage for AMPs because development of microbial resistance by gene mutation is less likely [3]. AMPs obtained from higher eukaryote organisms are also frequently referred to as ‘host defense peptides’, a term that emphasizes their additional immunomodulatory activities [2]. Traditionally, the search for novel AMPs involved the identification of active peptides from natural sources. Such studies are followed by the design of synthetic peptide Corresponding author: Vogel, H.J. ([email protected]).
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analogs for structure–function studies. De novo peptide design approaches have also been used, including highthroughput combinatorial library screening, structurebased modeling, predictive algorithms and the introduction of non-coded modifications to conventional peptide chemistry [4,5]. Furthermore, AMPs are being discovered through sequence searches for potential antimicrobial fragments within large proteins. In fact, several proteins have now been shown to exert direct antimicrobial activity themselves, further widening the scope of the structural frameworks for AMPs or antimicrobial proteins. Generally, two physical features are common for AMPs: a cationic charge and a significant proportion of hydrophobic residues. The former property promotes selectivity for negatively charged microbial cytoplasmic membranes over zwitterionic mammalian membranes whereas the latter facilitates interactions with the fatty acyl chains [6]. There are also a few anionic AMPs, such as dermcidin, although other biological activities seem to be more important for these peptides [7]. Many linear AMPs are unstructured in aqueous solution and require a membranous environment to adopt a stable amphipathic conformation [8]. Although the mode of action usually involves disrupting the integrity of the bacterial cytoplasmic membrane in many ways (Figure 1), other antimicrobial mechanisms have now been characterized that target key cellular processes including DNA and protein synthesis, protein folding, enzymatic activity and cell wall synthesis [9–11]. A comprehensive list of mechanisms utilized by AMPs is presented in Table 1. Membrane interactions remain important even for intracellular-targeting peptides because they must have a means of translocation. The cationicity of the AMPs also promotes interactions with negatively charged moieties on other biomolecules such as outer membrane lipids, nucleic acids and phosphorylated proteins. During an infection, a host animal may release multiple isoforms or structurally similar AMPs that act by distinct mechanisms to achieve an overall synergistic effect [12]. Furthermore, for some of the better studied AMPs, such as magainin and indolicidin, it has been found that they can have multiple modes of action, suggesting that they may work as ‘dirty’ antimicrobials that rely on a multiple-hit strategy to increase their efficiency and evade potential resistance mechanisms [3]. In this review, we aim to illustrate the wide diversity of structural properties demonstrated by AMPs as well as the growing number of ways in which they exert their activities.
0167-7799/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2011.05.001 Trends in Biotechnology, September 2011, Vol. 29, No. 9
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Toroidal pore Disordered toroidal pore Carpet model +
Membrane adsorption
Barrel stave +
+
+
+
+
Conformational change
Δd
Membrane thinning/thickening
+
0.2V
? Electroporation Charged lipid clustering Bacterial membrane ΔΨ
O Non-lytic membrane depolarization
+
+
H
+
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Anion carrier
Oxidized lipid targeting TRENDS in Biotechnology
Figure 1. Events occurring at the bacterial cytoplasmic membrane following initial antimicrobial peptide (AMP) adsorption. These events are not necessarily exclusive of each other. In the classical models of membrane disruption, the peptides lying on the membrane reach a threshold concentration and insert themselves across the membrane to form either peptide-lined pores in the barrel-stave model, solubilize the membrane into micellar structures in the carpet model, or form peptide-and-lipidlined pores in the toroidal pore model. In the revised disordered toroidal pore model, pore formation is more stochastic and involves fewer peptides. The thickness of the bilayer can be affected by the presence of the peptides, or the membrane itself can be remodeled to form domains rich in anionic lipids surrounding the peptides. In more specific cases, non-bilayer intermediates in the membrane can be induced; peptide adsorption to the membrane can be enhanced by targeting them to oxidized phospholipids; a peptide may couple with small anions across the bilayer, resulting in their efflux; the membrane potential can be dissipated without other noticeable damage; or conversely, in the molecular electroporation model, the accumulation of peptide on the outer leaflet increases the membrane potential above a threshold that renders the membrane transiently permeable to various molecules including the peptides themselves.
Recent structure–function relationship studies will be presented within the framework of the three AMP structural groups: a-helical peptides, b-sheet peptides and extended peptides (Figure 2) [13]. a-Helical AMPs The a-helical AMPs, such as magainin from the African clawed frog, are amongst the most intensely studied AMPs [14,15]. Numerous structure–function studies have indicated that membrane-bound a-helices with too large a hydrophobic surface become cytotoxic to mammalian cells [16]. Although these peptides are characterized by their structure, an extremely high propensity for the formation of the a-helix, which may present a more continuous hydrophobic surface, can become a contributing factor to cytotoxicity [17]. Indeed, peptides that remain antimicrobial but have low cytotoxicity require a membranous environment to induce proper folding and often have a proline- or glycine-induced
kink in the middle of the a-helix. The amphipathicity of the peptides is usually segregated along the axis of the a-helix such that the peptide can lie parallel to the membrane plane during the initial lipid interactions, with the charged side facing outward towards the phospholipid head groups and the hydrophobic side embedded into the acyl tail core. The properties of the a-helix tails sometimes influence the depth of membrane insertion and, in turn, the antimicrobial activity [18]. The lengths of the a-helices are usually sufficient to span the thickness of the membrane bilayers, with mismatches between the hydrophobic portion of the helices and the lipid acyl core strongly influencing the AMPs’ lateral and orientational motions in the membranes [19,20]. The peptides are thought to disrupt bacterial membranes through the formation of toroidal pores composed of loosely associated peptides with interdigitating phospholipid head groups among them [10]. Pore formation is often associated with a local change in 465
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Table 1. Different modes of action that have been described for AMPs Major target External proteins Outer surface lipids
Outer membrane proteins (Gram-negative)
Inner membrane
Integral membrane proteins Nucleic acids
Intracellular proteins
Specific target/mode of action Autolysin activation Phospholipase A2 activation LPS permeabilization (Gram-negative) Lipid II (peptidoglycan precursor)
Example peptides a Pep5, nisin Magainin 2, indolicidin Cecropin P1 Defensins, Nisin and other lantibiotics
Outer membrane protein I (OprI) LPS-assembly protein D (LptD) inhibition Outer membrane protein F (OmpF) Barrel-stave pore Detergent micellization Toroidal pore Disordered toroidal pore Membrane thinning/thickening Charged lipid clustering Non-bilayer intermediate formation Oxidized phospholipid targeting Anion carrier Non-lytic membrane depolarization
SMAP-29, CAP-18 Protegrin I peptidomimetic analogs HP(2–20) analog Alamethicin Dermaseptin, cecropin Magainin 2, melittin protegrin I Magainin analog, melittin PGLa, LL-37 Magainin analogs, Arg-rich peptides Gramicidin S Temporin L, indolicidin Indolicidin Bovine lactoferricin, daptomycin
Electroporation Proton translocation-related proteins DNA (general) DNA (covalent interaction) Branched DNA RNA (general) DnaK inhibition, GroEL chaperonin 20S proteasome, SH3-containing proteins
NK-lysin Clavanin A Buforin 2, tachyplesin, indolicidin Indolicidin WRWYCR Buforin 2 Pyrrhocoricin, drosocin, apidaecin PR-39
Refs [10] [10] [69] [34,35] [70] [71] [33] [72] [10] [10] [10] [31] [22] [21] [26] [73] [27] [49] [32] [74] [42] [28] [10] [50] [43] [10] [10] [46] [47]
a
Peptides listed are not limited to their associated modes of action here and may employ multiple mechanisms simultaneously.
membrane thickness [21]. Recent molecular dynamics (MD) simulations with a magainin analog and with melittin have suggested that a re-evaluation of this model is in order [22,23]. In the ‘disordered toroidal pore’, lipid molecules are still curved inwards, but pore formation is more stochastic and only one or two peptides are located near the center of the water-permeable pore, with more peptides positioned at the mouth of the pore on the external leaflet. As they translocate to the interior of the cell, the peptides remain mostly parallel to the bilayer plane, and only a small tilt is observed. Interestingly, the peptides undergo partial unfolding during translocation and their a-helicity is not always maintained. In other MD simulations, similar tilted orientations for a-helical peptides were observed and the notion of imperfect amphipathicity was introduced [24]. This refers to the presence of some polar or charged residues in the hydrophobic face of an AMP to pull lipid head groups into the membrane interior, thereby facilitating pore formation. A perfect hydrophobic face on a peptide would favor a fixed parallel orientation on a flat membrane surface. Imperfect amphipathicity is also a highlighted feature in the proposed interfacial activity model [25], which is based on a recent review of AMP literature. It closely resembles the disordered toroidal pore model, with the key difference of the interfacial activity model describing membrane disruption without a need for peptide self-assembly. The carpet model, where AMPs act more in a detergentlike manner to break off the membrane lipids into micellelike structures, has also been described. This model 466
appears to be applicable mostly to the high concentrations used in vitro, and is generally viewed as an extreme extension of the toroidal pore model [10]. An interesting membrane perturbing effect induced by some a-helical AMPs is that of lipid segregation, where peptide-induced clustering of anionic lipids in bacterial membranes causes slow leakage of intracellular contents and/or membrane depolarization [26]. Alternatively, the phase boundary effects between domains of differently charged lipids may compromise the overall membrane stability. Moreover, many bacterial membranes are mostly composed of phosphatidylglycerol and phosphatidylethanolamine lipids, where the latter does not like to form stable bilayers after peptide-induced demixing. In the case of Gram-positive bacteria such as Staphylococcus aureus, which have cytoplasmic membranes that are mainly composed of the negatively charged lipids phosphatidylglycerol and cardiolipin, lipid segregation is not expected and this is reflected in lower toxicity of some AMPs against these species [26]. The targeting of oxidized lipids represents a unique membrane-associated means for AMPs to enhance their activity. The a-helical AMPs temporin B and L intercalate more efficiently into membranes containing an oxidized phosphatidylcholine lipid, probably via Schiff base formation between the peptide amino groups and lipid aldehyde groups [27]. With the release of reactive oxygen species during phagocytosis, it is possible that lipid oxidation could increase bacterial membrane susceptibility to AMPs.
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(a)
LL-37
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Magainin 2
(b)
Bovine lactoferricin
Protegrin I Human β-defensin-3
(c)
RRWQWR Tritrpticin
Indolicidin TRENDS in Biotechnology
Figure 2. An overview of the major structural classes of antimicrobial peptides (AMPs). (a) a-Helical peptides, (b) b-sheet peptides and (c) extended peptides. All of these structures were solved by solution NMR spectroscopy in the presence of detergent micelles, except for the b-sheet peptides, which were studied in aqueous solution. Positively charged side chains are colored in blue, negatively charged side chains in red and remaining side chains in grey. PDB IDs: magainin 2, 2MAG; LL-37, 2K6O; bovine lactoferricin, 1LFC; protegrin 1, 1PG1; human b-defensin-3, 1KJ5; tritrpticin, 1D6X; indolicidin, 1G89.
Finally, the Phe-, His- and Gly-rich peptide clavanin A has a unique dual pH-dependent mode of action [28]. At neutral pH, it permeabilizes membranes like regular ahelical AMPs. At slightly acidic pH, clavanin A is still membrane disruptive. However, this does not occur via lipid interactions, but rather through inhibitory interactions with proteins involved in maintaining the pH gradient across the membrane. In contrast to most a-helical peptides, buforin II, a peptide derivative from histones of the Asian toad Bufo bufo gargarizans, does not cause membrane disruption [9] although its cell entry can be affected by membrane lipid composition [29]. After translocation, buforin II accumulates in the cytoplasm where it is known to exert its antimicrobial activity by adapting an extended or polyproline II conformation to accommodate interactions with nucleic acids [30]. b-Sheet AMPs The b-sheet AMPs include several b-hairpin peptides in addition to the defensin mini-proteins. Many of these peptides disrupt bacterial membranes via the formation of toroidal pores as observed for the porcine peptide protegrin I. This peptide forms oligomeric transmembrane bbarrels in anionic membranes, but forms b-sheet aggregates on the surface of cholesterol-containing membranes [31].
Additional evidence suggests that non-lytic mechanisms are also employed by certain b-sheet peptides. Although tachyplesin from horseshoe crabs is generally recognized as a membrane active peptide, it is also capable of binding to the minor groove of DNA, which may interfere with DNA–protein interactions [10]. The b-hairpin peptide bovine lactoferricin can act synergistically with other antimicrobial agents by affecting the transmembrane potential and proton-motive force, resulting in inhibition of ATPdependent multi-drug efflux pumps [32]. Following translocation, bovine lactoferricin localizes to the cytoplasm where it can also inhibit DNA, RNA and protein synthesis. A series of peptidomimetic analogs of protegrin I featuring stabilized b-hairpins are no longer membrane active, but employ a new bactericidal mechanism involving a specific protein target that affects outer membrane biosynthesis [33]. Although these peptide analogs are remarkably potent, their spectrum of activity is narrowed to Pseudomonas aeruginosa strains, which express the susceptible homolog of this b-barrel protein. Finally, although the human defensins are membrane active, some of them can also bind to the peptidoglycan precursor lipid II to inhibit cell wall biosynthesis enzymes in Staphylococci [34,35]. The b-hairpin or b-sheet conformations of these peptides are often stabilized by disulfide bridges between conserved Cys residues. However, these covalent bonds 467
Review do not appear necessary for antimicrobial activity. Linear derivatives of tachyplesin and lactoferricin retain antimicrobial activity while losing hemolytic activity [32,36]. Solid state NMR studies of tachyplesin and two linearized analogs, in which the four Cys residues were substituted by either Phe or Ala residues, show that all three peptides are positioned at the membrane interface and adopt similar bsheet conformations on anionic membranes [37]. However, this does not correlate with their activities: the Cys-to-Phe peptide is equally active as tachyplesin whereas the Cysto-Ala peptide is inactive. Peptide dynamics, rather than their structures, provide a reasonable explanation for these functional differences. Whereas the active tachyplesin peptide shows enough mobility in the liquid-crystalline phase of the membrane to disturb lipid packing, the inactive analog is immobilized on the membrane and stays highly aggregated. At 45 residues, human b-defensin-3 (HBD-3) is rather large as an AMP, containing an a-helical turn followed by a three-stranded antiparallel b-sheet. This scaffold is held together by three disulfide bonds in a defined pattern that distinguishes the b-defensins from the a-defensins. Nonetheless, rearranging the disulfide pattern in HBD-3 or reducing them does not alter the antimicrobial activity, nor does it abolish the overall formation of the native secondary structure elements [38,39]. Although antimicrobial activity may be unaffected, chemotactic activities are decreased for the analogs with non-native disulfide linkages. Interestingly, reducing the disulfides in HBD-1 creates a flexible unstructured peptide with improved activities against Candida albicans and some Gram-positive bacteria [40]. The presence of reduced HBD-1 could be of great physiological relevance in the anaerobic environment of the colon. Extended AMPs Extended AMPs, which do not fold into regular secondary structure elements, often contain high proportions of certain amino acids, specifically Arg, Trp or Pro residues. The His-rich salivary histatin peptides are sometimes considered to be extended AMPs, however they are not bacteriostatic nor bactericidal despite being fungicidal and antiparasitic [41]. Many of the extended peptides are not membrane active. The Pro-rich insect-derived pyrrhocoricin, drosocin and apidaecin peptides penetrate across the membrane and exert their antimicrobial activities by making interactions with intracellular proteins such as the heat-shock protein DnaK and GroEL to inhibit the DnaK ATPase activity and chaperone-assisted protein folding, respectively [10]. Trp and Arg side chains are prominent in peptides with less than 15 residues [42]. Two of the shortest active peptides include RRWQWR and RAWVAWR, which have been identified as the active antimicrobial fragments from bovine lactoferricin and human lysozyme, respectively. In addition, combinatorial peptide library screening has identified the hexameric sequence RRWWRF as having potent broad spectrum activity [4]. Although these peptides can fold into defined amphipathic molecules in the presence of a membrane, they are not membrane active and, instead, 468
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they accumulate in the cytoplasm. The hexapeptide WRWYCR, which was identified as a DNA-repair inhibitor that binds to Holliday junctions, was subsequently shown to have broad bactericidal activity [43]. Given the sequence similarity of this peptide to those mentioned above, their efficacy may be due to this mechanism, where trapping of the replication fork prevents recombination and DNA repair. The 13-residue Arg- and Trp-rich tritrpticin and indolicidin peptides from porcine and bovine leukocytes, respectively, can cause membrane leakage [42]. In a membranous environment, the structures of both peptides feature multiple Pro-induced turns that give them amphipathic boat-like conformations. These peptides are sufficiently small that simple residue substitutions can have large consequences for their structural and functional properties. Replacing both Pro residues with Ala in tritrpticin transforms it into an a-helical peptide with slightly improved membrane leakage and antimicrobial activity, but also higher cytotoxicity [44]. The role of the two Pro residues is distinct, with Pro5 having more of a structural role preventing the induction of a-helical structure. Ironically, the characteristic Arg and Trp side chains do not seem an optimal choice of residues for tritrpticin. In analogs with Arg-to-Lys and Trp-to-Phe substitutions, the antimicrobial activity is maintained and the slight hemolytic activity of tritrpticin is abolished. The reduced hydrogen bonding capabilities of Lys compared to the Arg guanidinium group seem to increase membrane selectivity for the peptide. Replacement of the three Trp residues with Phe created a tritrpticin variant that was no longer able to adopt a single well-defined conformation in a membrane [44]. Interestingly, this peptide required a few hours to take effect whereas native tritrpticin completes bacterial killing in less than one hour [45]. The fact that multiple mechanisms are in play is further substantiated by the synergistic effects observed between the Trp-to-Phe and membrane active Pro-to-Ala peptides [45]. The Pro- and Arg-rich porcine PR-39 peptide interacts with cytoplasmic proteins such as the 20S proteasome and SH3-domain proteins, and it is believed that this contributes to its antimicrobial mode of action [46,47]. Indolicidin has been a template for many analogs with improved biological properties, the most successful one being omiganan, which was developed by Migenix (renamed to Biowest Therapeutics) and has entered phase III clinical trials [48]. A number of contributing mechanisms may explain the efficiency of indolicidin. This includes the activation of phospholipase A2 secreted by human lacrimal fluid to hydrolyze anionic membranes [10] or a direct membrane action either through lipid segregation, the targeting of oxidized phospholipids or acting as a small anion carrier across the membrane [27,49]. Moreover, at the intracellular level, both covalent and noncovalent DNA–indolicidin interactions can interfere with DNA-binding enzymes and induce filamentation in Escherichia coli [50]. It is unclear how the short and unique sequence of indolicidin, which includes five Trp residues and three Pro-induced turns, can exhibit all these activities and which of these proposed mechanisms contributes most to its activity in vivo.
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Protein-derived antimicrobial fragments and antimicrobial proteins AMPs can be derived from large proteins. In addition, several cationic proteins have been discovered to possess direct nonenzymatic antimicrobial activity themselves, some of which are shown in Figure 3. The primary function of these antimicrobial proteins is usually immunity-related, therefore they show significant functional overlap with AMPs (Table 2). Lactoferrin (Lfin) is a multifunctional 80 kDa glycoprotein present in biofluids, most notably in milk and neutrophil granules. The protein is involved in several immunoregulatory processes that primarily take advantage of its strong antimicrobial properties. This activity is due in part to its ability to sequester iron, which prevents bacterial growth. However, Lfin also has an iron-independent mechanism arising from direct interactions with bacterial cell components [51]. Three cationic antimicrobial fragments have been identified in different parts of the Lfin sequence: lactoferricin, which is a naturally occurring pepsin digestion product, lactoferrampin and kaliocin (Figure 3a) [18,32,52]. The structural differences for the isolated peptides and the corresponding regions in Lfin are significant; in particular, bovine lactoferricin and kaliocin form amphipathic b-hairpin peptides, whereas lactoferrampin requires a membraneous environment to adopt its native a-helical structure. The intact protein and the peptides probably kill bacteria in a different manner, especially lactoferricin, which is substantially more active than intact Lfin.
[(Figure_3)TD$IG]
Whether they are naturally released proteolysis products or designed synthetic peptides, many AMPs have been derived from proteins such as casein [53], lysozyme and ovotransferrin [54], myoglobin and hemoglobin [55], histones [9], growth factors [56], and thrombin [57]. Thus, scanning the proteome for related sequences with appropriate combinations of cationic and hydrophobic residues may uncover additional AMPs. Some of these proteins such as lysozyme and histones already have intrinsic antimicrobial activity and, as is the case for Lfin, their peptide fragments are expected to have different modes of action [58]. Recently, more than 20 human chemokine proteins have been shown to be directly antimicrobial, with macrophageinflammatory protein-3a (MIP-3a) possessing the most potent and broadest spectrum of activity (Figure 3b) [59]. In the case of the chemokine CCL28, the antimicrobial mechanism has been identified as membrane permeation [60]. Conversely, a few defensins are now known to possess chemotactic activity [61]. A common and unifying feature of the antimicrobial chemokines is the presence of a large uninterrupted cationic surface. Some chemokines tend to dimerize under physiological conditions, further extending the cationic surface [62]. For example, HBD-3 has a stronger tendency to dimerize, which may contribute to its high salt-insensitive activity compared to other b-defensins [63]. The C-terminal a-helices of several antimicrobial chemokines have multiple positively charged residues and when synthetic peptides of this region are studied, they behave like membrane-active a-helical AMPs [64].
(a)
(b)
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Figure 3. The structures of immunity-related proteins with direct antimicrobial activity and antimicrobial fragments. (a) Human lactoferrin (PDB ID 1FCK) with segments corresponding to its antimicrobial fragments: lactoferricin in blue, kaliocin in red and lactoferrampin in yellow (left panel). The electrostatic potential surface plot of lactoferrin is shown in the right panel. (b) Macrophage inflammatory protein-3a (PDB ID 2JYO) with the MIP-3a51–70 peptide segment colored in blue (left panel). The electrostatic potential surface plot of the monomeric unit of MIP-3a is shown in the middle panel and the same plot for the MIP-3a dimer is in the right panel, showing the stronger cationic surface around the self-associating a-helices.
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Table 2. A selection of human AMPs and antimicrobial proteins highlighting their multifunctionality in the immune system Main sources
Indirect antimicrobial and additional functions a
Refs
AMPs LL-37
Leukocytes, epithelia
[61]
Defensins
Leukocytes, epithelia
Dermcidin Histatins Lactoferricin
Skin Saliva Digested milk protein
Endotoxin neutralization, chemotaxis, reactive oxygen species (ROS) formation, wound healing, mast cell degranulation Endotoxin neutralization, opsonization, chemotaxis, ROS formation, wound healing, complement inhibition Chemotaxis Wound healing Endotoxin neutralization, complement inhibition, transcription factor
Antimicrobial proteins Chemokines Neutrophil serprocidins (azurocidin, cathepsin G, elastase) Bactericidal/permeability increasing protein Lipocalins Lysozyme Lactoferrin
Neutrophils, secretory fluids Secretory fluids, neutrophils Secretory fluids, neutrophils
RNAse 7 Psoriasin Histones Prion protein
Skin Skin Nuclei Brain, other tissues
Leukocytes Neutrophils Neutrophils
Chemotaxis Protease, ROS formation, opsonization, macrophage activation Endotoxin neutralization Siderophore sequestering Peptidoglycan digestion Iron sequestering, endotoxin neutralization, phagocytosis stimulation Ribonuclease Neutrophil activation, ROS formation Chromatin regulation Unknown
[61] [7] [75] [32]
[59] [61] [61] [76] [61] [61] [77] [77] [78] [79]
a
These are usually secondary functions for AMPs and primary functions for antimicrobial proteins.
This property may explain the bactericidal properties of several intact chemokines; however, some of the a-helical peptides do not retain activity and, furthermore, some chemokines do not have cationic C-terminal regions despite displaying activity. For the platelet chemokine neutrophil activating protein-2 (NAP-2), the proteolytic removal of the two C-terminal Ala-Asp residues leads to a drastic increase in the antimicrobial potency. This was attributed to the negatively charged Asp side chain interfering with a positive patch on the protein surface that includes side chains from the C-terminal a-helix as well as the b-sheet loops [65]. Concluding remarks Widely different structural scaffolds seem to support the activity of AMPs and proteins, with amphipathicity remaining as the only overarching physical property. A combination of a well-defined cationic region together with an imperfect hydrophobic surface area frequently seems to result in broad spectrum activity. In addition, the dynamic properties of these peptides can affect their biological properties, with structural rigidity generally hindering activity. Initially, AMPs were considered as a uniform group of molecules that ‘punch holes’ in bacterial membranes. In recent years, it has become apparent that their mechanism of action is much more complex and diverse. Current research efforts now also include antimicrobial fragments from intact proteins and proteins with direct antimicrobial activity, forcing structural biology researchers to reevaluate the common properties within this expanding family. Clearly, host defense systems have developed a broad arsenal and use a multi-pronged strategy to combat invading pathogens. Many AMPs seem to be capable of killing bacterial cells in multiple ways, making it a difficult task to unravel all the molecular events that occur when a peptide encounters a 470
bacterial cell. Consequently, the design and prediction of peptide analog characteristics is not straightforward and all peptides and analogs need to be studied on a case-by-case basis. Apart from their biological activities, other aspects need to be considered when developing these peptides for systemic use, including in vivo stability [66], side effects and production costs. Based on our current understanding of the structure–function relationships for different classes of AMPs, some non-peptide and peptidomimetic compounds have been developed and have advantageous properties because they combine stability with high antimicrobial potency [67,68]. Overall, detailed structure–function and biophysical studies can rationalize why a certain peptide is more active or behaves differently from a related peptide or an analog. Such comprehensive understanding will be essential to guide the design of better AMPs. Acknowledgments Research on AMPs in the H.J. Vogel laboratory is supported by a grant from the Novel Alternatives for Antibiotics Program of the Canadian Institute for Health Research. H.J.V. is the holder of a Scientist award from the Alberta Heritage Foundation for Medical Research.
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