Reviews Cite This: ACS Chem. Biol. 2018, 13, 844−853
Antimicrobial Metallopeptides Jessica L. Alexander,† Zechariah Thompson,† and J. A. Cowan* Evans Laboratory of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, United States ABSTRACT: Antimicrobial peptides are short amphipathic peptides that are produced by the innate immune system in order to protect a host from pathogens. They have been shown to have broad-spectrum antimicrobial activity toward Gram-positive and Gram-negative bacteria, as well as antifungal, antiprotozoan, and antiviral activity. These peptides are able to exert their activity through a variety of mechanisms that include inhibiting DNA and RNA replication, inhibiting protein synthesis, permeabilizing the cell membrane, disrupting proton and ion transmembrane gradients, and inhibiting cell wall biosynthesis. Certain antimicrobial peptides are able to utilize metals to modulate their activity through structural changes upon metal binding, metal sequestration, and redox chemistry. This work aims to provide a review of the current literature regarding the influence of metals on the activity of antimicrobial metallopeptides and their uses in drug delivery and the treatment of implant-associated infections.
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INTRODUCTION Overview of Antimicrobial Peptides. Antimicrobial peptides (AMPs) are naturally occurring peptides that play an important role in the innate immune system. These peptides are found in many different types of organisms, including mammals, invertebrates, plants, and insects.1 Although most AMPs range from 10 to 100 amino acids in length, others such as calprotectin and psoriasin are larger and form quaternary structures. Additionally, AMPs have a relatively high proportion of hydrophobic and positively charged residues arranged in an amphipathic design. Although most antimicrobial peptides are positively charged, anionic peptides have also been identified.2 Antimicrobial peptides can be classified into four groups according to their structure, β-sheet, α-helix, loop, and extended structures. Targets of Antimicrobial Peptides. Antimicrobial peptides are able to inhibit many pathogens, including Gramnegative and Gram-positive bacteria and fungi. Additionally, some antimicrobial peptides have been shown to have anticancer or antiviral activity, such as indolicidin that has activity toward HIV.3 These peptides were originally thought to target the cell membrane through a variety of mechanisms that include cell membrane permeabilization and disruption of either proton or ion transmembrane gradients or disruption of cell membrane biosynthesis.4,5 Although many peptides are thought to exert their activity through binding to the cell membrane, numerous studies have demonstrated that these peptides can also bind to additional intracellular targets that include DNA, RNA, and proteins in any of the important cellular compartments.4,6 A general summary of common modes of action is displayed in (Figure 1). In addition to their activity toward pathogens, antimicrobial peptides have important functions in the innate immune system and have been associated with several disease states, such as cystic fibrosis, psoriasis, and inflammatory bowel disease.7,8 Following secretion, AMPs are able to take on a multitude of © 2018 American Chemical Society
functions, such as antimicrobial activity, cell activation, apoptosis, and modulation of the immune response.7 Antimicrobial Peptides as Novel Antibiotics. The large increase in antibiotic resistance, combined with the decrease in novel antibiotic therapies, could lead to a “pre-antibiotic” era, where no antibiotics are available to treat infection.9 Consequently, it is vital to develop new therapies, and antimicrobial peptides are an attractive option due to their ease of synthesis and broad-spectrum activity. Although they represent an attractive avenue to explore, novel antibacterials are typically limited in their chemical diversity, and selecting a target that is not prone to resistance makes them challenging from a drug design perspective.10 Furthermore, bacteria are able to reduce their effectiveness by altering their membrane composition, by efflux through energy-dependent pumps, and by cleavage with proteases.11 Despite these limitations, several antimicrobial peptides have shown promising activity and have been accepted into clinical trials, including AMP hLF1-11 (a lactoferrin-derived peptide), pexiganan, and omiganan.12−14 While there is a large amount of literature regarding the function and mode of action of antimicrobial peptides, few studies have investigated the effect of metals on the activity of peptides. Of the over 2000 antimicrobial peptides identified, only a handful of them have been demonstrated to utilize metals to modulate their activity. Given that a large portion of proteins require metal ions for activity, it would not be surprising if many more antimicrobial peptides can be regulated through metal ions. This review serves to provide an overview of current literature regarding the impact of metal ions on the activity of antimicrobial peptides through metal sequestration, activation through structural change, use of metals for Lewis Received: November 20, 2017 Accepted: February 1, 2018 Published: February 1, 2018 844
DOI: 10.1021/acschembio.7b00989 ACS Chem. Biol. 2018, 13, 844−853
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Figure 1. Cell diagram depicting modes of action for antimicrobial peptides.
acid and redox catalysis, and engineered derivatives for drug delivery and treatment of implant-associated infections.
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METAL-ACTIVATED ANTIMICROBIAL PEPTIDES Several AMPs require metal ions such as Zn2+, Na+, and Mn2+ for antimicrobial activity. In some cases, coordination of the metal allows for preorganization of the AMP and subsequent ligand binding mediated through charge−charge or metal− ligand interactions.15 These peptides have been proposed to bind to the microbe in the presence of their cofactors, ultimately leading to destabilization or permeabilization of the cell wall.16 Other evidence has shown an alternative mechanism in which the metal-bound AMP binds to molecules important for biosynthetic pathways.16−18 Bacitracin A. Bacitracin A, a cyclic nonribosomally synthesized peptide produced by Bacillus subtilis and Bacillus licheniformis,19,20 has been shown to promiscuously bind a variety of divalent metals.21 In one particular case, a synthetic Mn-bacitracin complex showed enhanced inhibition of antimicrobial growth toward Gram-positive S. aureus and Enterococcus spp.22 This metalloantibiotic also displayed potent superoxide dismutase activity even in the presence of EDTA and BSA, suggesting it could act under oxidative stress. Under physiologically relevant conditions, bacitracin has been shown to bind undecaprenyl pyrophosphate, which is involved in transporting intermediates required for cell wall biosynthesis.18 Binding to the pyrophosphate group was of highest affinity in the presence of zinc. These observations suggest a two-step mechanism of action. First, it confirms the requirement of divalent ions for antimicrobial activity. Second, it shows that bacitracin A binds the undecaprenyl pyrophosphate ligand at concentrations comparable to observed minimal inhibitory concentrations (MICs), suggesting the primary mode of action for antimicrobial activity to involve undermining the bacterial envelope by interfering with this key biosynthetic pathway. Recently, a 1.1 Å crystal complex of Zn-bacitracin and undecaprenyl pyrophosphate has been solved that shows the antibiotic wrapped around the ligand target and coordinating to both Zn2+ and a previously unknown Na+ ion (Figure 2).15 Zn2+ coordinates with an octahedral geometry with donor atoms from the N-terminus of the peptide, Glu4, and thiazoline
Figure 2. A 1.1 Å crystal structure of ternary complex of bacitracin A. The peptide is complexed with Zn2+ (depicted blue), Na+ (depicted purple), and geranyl-pyrophosphate (pyrophosphate group in redorange and geranyl group displayed white in color). Hydrophobic residues encapsulate the geranyl group closely with a distance of 3.5−4 Å. Ligand−metal and residue−metal interactions are depicted as dashed lines (PDB 4K7T).
nitrogen. His10 is absent from the coordination sphere, which had been a key ligand noted in the binary zinc−bacitracin complexes solved earlier.23−25 Instead, Zn2+ coordinates two oxygen atoms from the pyrophosphate and an additional water molecule. The Na+ ion coordinates antipodal to Zn2+, with undecaprenyl pyrophosphate appearing to neutralize the negatively charged ligand and organize the C-terminus of the peptide. The hydrophobic leucine and isoleucine residues appear to sequester the diprenyl chain, preventing hydrolytic release of the lipid and inhibiting biosynthesis of the cell wall. Dermcidin. Dermcidin is an antimicrobial peptide secreted from eccrine sweat glands and was shown to be proteolytically processed by mass spectrometry amino acid sequencing. Two fragments, DCD-1 and DCD-1L, showed potent antimicrobial activity against E. coli, Enterococcus faecalis, and S. aureus with 845
DOI: 10.1021/acschembio.7b00989 ACS Chem. Biol. 2018, 13, 844−853
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ACS Chemical Biology MICs measuring ∼1 μg/mL,26 with the latter peptide having an additional leucine residue at the C-terminus. Circular dichroism (CD) and oriented-CD demonstrated that the peptides adopt an α-helical conformation in the biomimetic solvent, trifluoroethanol.16,27,28 Oligomerization was also observed for DCD-1L and other similar synthetic peptides, which compares well to SDS analysis of human sweat and suggests that these peptides could oligomerize in vivo.17,29 With the high concentrations of divalent cations in human sweat, particularly Zn2+, it was hypothesized that these divalent ions could be required for its function. DCD-1L oligomerization was found to be more stable in the presence of Zn2+ and is promoted by increasing the hydrophobicity of the solvent.16 Electrophysiology studies also suggested that following oligomerization, these peptides formed potent ion channels inside the membrane.16,30 A 2.5 Å crystal structure revealed more mechanistic information concerning the physiological relevance of Zn2+, as well as the requirement for oligomerization for function.30 The crystal structure, displayed in Figure 3, shows that the
functional model has been proposed that involves the cationic N-terminal region of the peptide binding the membrane electrostatically, facilitating its insertion into the membrane, while the anionic C-terminal region floats on the surface of the phospholipid bilayer.16,31 An extension of this model suggests that the C-terminal region folds back on the N-terminal helix creating a hairpin-like structure. Another model suggests that upon interacting with the bilayer, the homotrimeric helix inserts itself so that it is tilted with respect to the plane of the bilayer, facilitating disruption of the bacterial membrane. Dermcidin can then self-assemble into higher oligomeric states, which is facilitated by Zn2+ metal coordination.30,32 Clavanin A. Clavanin A (VFQFLGKIIHHVGNFVHGFSHVF-NH2) is a 23-residue antimicrobial peptide isolated from the tunicate invertebrate, Styela clava, which adopts an αhelical conformation in a hydrophobic environment.33 The peptide shows broad-spectrum antimicrobial activity toward Gram-negative and Gram-positive bacteria, is able to maintain its activity up to 0.3 M NaCl, and also has enhanced activity at lower pH34,35 where clavanin A exerts its activity in E. coli through membrane permeabilization.34 Conversely, calcein leakage assays have demonstrated that clavanin A is able to permeabilize liposomes at neutral pH but not at pH 5.5.35 This study also showed that clavanin A disrupted the transmembrane proton and ion gradients at low pH in Lactobacillus sake.35 These two experiments show that clavanin A shows two distinct mechanisms of action toward the membrane at different pH. However, due to the absence of cellular data for L. sake membrane permeabilization, the exact mechanism remains unclear. Recently, clavanin A was also shown to have enhanced activity in the presence of Zn2+ due to chelation with His17 and His21.36 This effect was not observed in E. coli derivatives lacking zinc-transporter proteins.36 Furthermore, Zn2+ appears to affect the localization of the peptide. Clavanin A is able to locate intracellularly within 1 h, while most of the Zn2+-bound peptide remains at the membrane.36 Given that Zn2+−clavanin A is able to cleave plasmid DNA, the peptide could potentially target genomic DNA.36 The ability of the peptide to induce membrane depolarization is also enhanced with zinc, but the ability to permeabilize the cell membrane or cause potassium leakage is not affected.36 These assays were all performed at pH 5.5 and contradict prior studies demonstrating membrane permeabilization at low pH, likely due to the presence of Zn2+. Given the difference in the mode of action of clavanin A at neutral pH, the peptide could also show a difference in activity at neutral pH when bound to zinc.
Figure 3. A 2.5 Å crystal structure of human antimicrobial peptide dermcidin forming a hexameric membrane channel. Zn2+-coordinating side chains depicted as lines with Zn2+ ions shown as gray spheres. Channel formation occurs through trimerization of antiparallel dimers, differentiated by their red and blue colors (PDB 2YMK).
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peptide dimerizes into an antiparallel α-helical conformation that is stabilized by Zn2+ at the C- and N-termini, tetrahedrally coordinating Glu5, Glu9, His38′, and Asp42′. These dimers form a homotrimer following insertion into the bacterial membrane and create an ion channel. By analysis of conductivity measurements, the peptide was shown to form ion channels only in the presence of Zn2+.30,31 Substituting His38 with Ala abolishes channel formation, suggesting that formation of the channel is promoted by coordination of Zn2+.16 From these observations, it has been hypothesized that these peptides exist in a complex equilibrium of monomers and dimers that aggregate on the cell surface, which is facilitated by the acidic conditions of sweat and divalent Zn2+. While oligomerization and Zn2+ ions are crucial for its antimicrobial activity, the exact way in which dermcidin associates with and inserts itself into the bacterial membrane remains unclear. A
METAL SEQUESTERING ANTIMICROBIAL PEPTIDES Transition metals are essential nutrients for all pathogens. They show a variety of functions as cofactors with proteins, including structural stabilization, regulating protein activity, and redox chemistry.37 Many of these proteins are involved in critical cellular processes such as mediation of electron transport and catalysis. In order to prevent infection from pathogens, mammals have developed mechanisms that restrict the availability of these metals, a process referred to as nutritional immunity.38 Furthermore, hosts are also able to secrete antimicrobial peptides including microplusin, psoriasin, and calprotectin that utilize metal sequestration as a means to inhibit bacterial growth during an infection.39 846
DOI: 10.1021/acschembio.7b00989 ACS Chem. Biol. 2018, 13, 844−853
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ACS Chemical Biology Microplusin. Microplusin is an antimicrobial protein that is found in the hemocytes, ovaries, and fat bodies of ticks.40−42 It contains six cysteine residues that form three disulfide bonds and forms five α-helices with disordered N- and C-termini (Figure 4).40,43 Microplusin has shown antimicrobial activity
Figure 5. Crystal structure of (a) psoriasin in the Ca2+- and Zn2+bound state and (b) the Zn2+-binding site (PDB 3D0Y). The individual subunits are blue and purple. The Ca2+ ions are yellow, the Zn2+ ions are green, and the residues coordinating Zn2+ are labeled.
Figure 4. Solution NMR structure of microplusin from Rhipicephalus microplus (PDB 2KNJ). The sites that are proposed to interact with copper are highlighted in magenta and include Glu16, Leu17, Ile20, Lys71, Ile73, His74, Asp75, Ala77, Thr78, Ala79, and Asp81.
toward several Gram-positive bacteria, with the exception of S. aureus, while showing no activity toward Gram-negative bacteria, which may be a reflection of the physiological levels of copper present in each microbe.43 This protein also shows antifungal activity with MICs as low as 0.09 μM for Cryptococus neoformans.43,44 Addition of copper reverses the inhibitory effect of microplusin toward Micrococcus luteus and C. neoformans, suggesting that it functions by sequestering copper from pathogens.43,44 This mechanism of antimicrobial activity is also supported by experiments that show that microplusin negatively affects respiration in M. luteus, most likely as a result of the removal of metal from heme-copper terminal oxidases. In C. neoformans, microplusin inhibits melanization and the formation of the polysaccharide capsule, both of which require copper-dependent enzymes.44 Psoriasin. Psoriasin is a protein that is homologous to the S100 family of Ca2+-binding proteins. It recruits CD4+ Tlymphocytes and neutrophils during the immune response and is up-regulated in the presence of calcium, in psoriasitic epidermis, and in differentiating keratinocytes.45,46 The crystal structure of Ca2+-bound psoriasin shows the presence of a Zn2+binding site formed by three histidines and an aspartate residue, analogous to typical coordination sites in metalloproteases (Figure 5).47,48 The antimicrobial activity of psoriasin has been shown to be selective for E. coli and its activity is reduced when it is prebound with zinc, suggesting that it exerts its activity by sequestering zinc from the bacteria.49 Furthermore, psoriasin is secreted from keratinocytes, presumably to protect the skin. Treatment with antibodies specific for psoriasin results in increased levels of E. coli on the skin,49 suggesting that the physiological relevance of psoriasin is to protect the skin from E. coli. Calprotectin. Calprotectin is another member of the S100 family of proteins that forms a heterodimer, with His3Asp and His6 metal-binding sites at the dimer interface (Figure 6).50−52 The His3Asp site binds zinc with high affinity and manganese with much lower affinity, while the His6 site can bind both manganese and zinc with high affinity (Figure 6).52−54 The zinc
Figure 6. Crystal structure of calprotectin bound to Ca2+, Na+, and Mn2+ (PDB 4XJK). (a) Calprotectin α2β2 tetramer. (b) αβ dimer of calprotectin with the (c) His3Asp and (d) His6 metal-binding sites labeled. Ca2+ ions are shown in yellow, Na+ ions in magenta, and Mn2+ ions in cyan.
ion is able to displace manganese from the His6 site, suggesting a preference for zinc-binding over manganese,55 while Zn2+ competition experiments with the fluorescent zinc sensor HNBO−DPA revealed sub-picomolar affinity for Zn2+ binding to calprotectin, so the stability constant could not be determined within reasonable error.51 Metal-bound calprotectin is also stable to proteolysis, and mutation of His103 or His105 in the His6 site led to decreased activity, presumable due to a weaker affinity for Zn2+.51 This suggests that His6 coordination contributes to both the stability and activity of calprotectin. Calprotectin’s activity and metal binding is also enhanced in the presence of calcium, which improves the affinity for both zinc and manganese.53,55,56 This calcium dependence suggests that calprotectin may exist in a low-affinity state inside the cell, which can undergo a slight conformational change in the extracellular space at the site of infection, where calcium levels are higher, to bind zinc or manganese more tightly. Several studies have demonstrated that calprotectin uses a sequestration mechanism for zinc and manganese ions to inhibit bacteria. Calprotectin has been found in S. aureus abscesses, and mice lacking calprotectin have increased levels of metal and S. aureus.56 Media treated with calprotectin prior to inoculation with S. aureus inhibited its growth, and the effect was reversed with the addition of manganese and zinc, suggesting that calprotectin inhibits bacterial growth in 847
DOI: 10.1021/acschembio.7b00989 ACS Chem. Biol. 2018, 13, 844−853
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ACS Chemical Biology tissues.56 Furthermore, calprotectin increases superoxide levels in S. aureus and decreases superoxide dismutase activity, while mutants unable to bind zinc or manganese do not display this behavior.50,54 Additionally, calprotectin’s role in the innate immune response against Klebsiella pneumoniae has been demonstrated. Mice infected with K. pneumoniae have increased levels of calprotectin, while mice lacking calprotectin showed increased organ damage and reduced survival rates, and their macrophages were unable to phagocytose the bacteria.57 In broth dilution assays, calprotectin’s activity against K. pneumoniae was abolished in the presence of zinc, suggesting it targets K. pneumoniae by sequestering zinc from the bacteria.57 Although zinc-binding has been demonstrated to be important for the antimicrobial activity of calprotectin, mutation of the manganese-binding site abolishes its broad-spectrum activity, suggesting that manganese sequestration may be more critical than Zn2+ sequestration for host defense against pathogens.50 Due to the different requirements for transition metals for various bacteria, calprotectin may bind both manganese and zinc in order to chelate either metal, depending on the type of infection.
infected rat models. The mechanism of action appears to be through disruption of the bacterial membrane, as evidenced by SEM and β-galactosidase leakage. Lactoferrin has also been successfully appended to gold nanoparticles and displayed enhanced activity against both S. aureus and E. coli strains as opposed to the free peptide in solution.61 These enhanced activities appear to be a consequence of the high local concentration of immobilized AMP on the nanoparticle surface, facilitating substantially greater contact of the AMP with bacterial membranes. Silver nanoparticles have intrinsically been shown to display broad-spectrum antimicrobial activity against bacteria, fungi, and viruses, mediated mainly by the production of ROS.59,62 The conjugation of AMPs to silver nanoparticles has been shown to display a synergistic effect when tested against Gramnegative and Gram-positive organisms. One group covalently attached daptomycin, a lipo-antimicrobial peptide marketed under the name Cubicin, to silver nanoclusters and demonstrated improved activity against S. aureus when compared to treatment with a nonconjugated mixture of daptomycin and silver nanoclusters.63 This system was shown to effectively damage the bacterial membrane and DNA, while simultaneously generating ROS as evidenced by damage to genomic DNA and lipid peroxidation assays, respectively. Another group appended an isolated AMP from frog skin, Odorranain-A-OA1 to silver nanoparticles and demonstrated enhanced antimicrobial activity and low cytotoxicity to mammalian cells.64 Interestingly, polymyxin B was attached to silver nanoparticles and exhibited a 3-fold enhanced activity against P. aeruginosa and Vibrio f luvialis, two common multidrug resistant strains prevalent in hospital-acquired infections.65 Moreover, these polymyxin-B-capped silver nanoparticles were able to effectively resist biofilm formation after being electroplated to the surface of surgical blades, as evidenced by expanded zones of inhibition on agar plates. As evidenced by these studies, conjugating AMPs to metal nanoparticles appears to be a conventional strategy by promoting synergy, while simultaneously combining their modes of action, thereby decreasing the likelihood of developing resistance. Engineering and Functionalization of AMPs to Titanium Surfaces. Expanding on the concept of attaching AMPs to metal surfaces, an interesting approach has been reported that involves attachment of these antimicrobials to titanium-binding peptides, which can aid in preventing implant failure and the development of sepsis. These peptides contain a titanium binding motif that can adhere to the desired titanium alloy, in addition to the AMP of choice.66 To ensure retention of antimicrobial properties by the AMP, a rigid or flexible linker can be added to prevent deleterious interactions between the peptide and the titanium-binding motif. Recently, one group engineered a system with a triglycine linker that displayed modest activity in solution. However, following attachment of these peptides to titanium surfaces, bacterial adhesion was severely abated.67 Moreover, these peptides were also stamped to the titanium surfaces in a variety of patterns, and following fluorescent staining, bacterial growth was only exhibited in the nonstamped regions of the titanium surface. Another group utilized a derivative of histatin, JH8194, and appended it to a titanium-binding peptide via a flexible glycine-rich (GGGGS) or a rigid proline-rich (PAPAP) linker.68 It was shown through LIVE/DEAD cell staining techniques that the flexible linker resulted in reduced
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ENGINEERED ANTIMICROBIAL PEPTIDE DERIVATIVES AND THEIR APPLICATIONS Antimicrobial peptides are attractive candidates for combating antibiotic resistance as their broad-spectrum activity and relatively small size makes them attractive for drug development. However, their poor resistance to proteolysis, potential toxicity at therapeutic concentrations, and lack of selectivity in vivo require further fine-tuning to ensure that they are appropriately targeted, can be effectively administered, and display improved efficacy in vivo to be considered as viable therapeutics. Currently, several groups are interested in conjugating AMPs and their derivatives to nickel, silver, or gold nanoparticles or nanotubes for delivery purposes. Other avenues for use include the coupling of AMPs to metal interfaces, commonly used in implants or prosthetics, to combat implant-associated infections. This burgeoning multidisciplinary field of biomaterials and antimicrobial therapeutics sheds light on their development and application toward novel antibiotics. Antimicrobial Peptides and Nanochemistry. Recent developments in nanochemistry, namely, surface modifications of nanoparticles and nanotubes, has enabled immobilization of small molecules and AMPs onto metal nanoparticles for use in combating infection in implants or as potential delivery systems. The conjugation of LL-37, a human cathelicidin, to magnetic nickel nanoparticles at threshold polymer concentrations yielded a system capable of preventing proliferation of E. coli.58 Addressing implant-associated infections at the site of prosthetic placement has be explored by conjugating AMPs to the surface of TiO2 nanotubes for use in implants. Several groups have utilized HHC36 and Tet213 AMPs to develop systems that are amenable for broad-spectrum use, exhibit low toxicity, and show steady release of peptide from the coated surface.59 Gold nanoparticles conjugated with both thioalkenes and the lipo-antimicrobial peptide surfactin exhibit broad-spectrum activity against clinically relevant bacterial strains.60 Through covalent attachment of both molecules, the gold nanoparticle system exhibited an ∼80-fold lower MIC than surfactin alone, while also exhibiting improved wound healing in MRSA848
DOI: 10.1021/acschembio.7b00989 ACS Chem. Biol. 2018, 13, 844−853
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determined by mass spectrometry.73,74 While both histatins are able to bind up to four atoms of copper and nickel, only one binding site for Zn2+ was observed, which corresponds to the HEKHH sequence.73 In Candida albicans, histatin 5 is localized in the mitochondria and inhibits respiration through production of ROS, which is also correlated to the inhibition of cell growth.75 The formation of ROS is also inhibited in the presence of a radical scavenger such as L-cysteine or TEMPO, suggesting that histatin 5 is targeted to the mitochondria in yeast and induces ROS in a critical step that promotes cell death.75 Induction of ROS formation in yeast from histatin 5 is likely due to the presence of the Cu-ATCUN motif. Many studies have investigated the importance of the ATCUN motif for ROS generation. An analogue of histatin 5 that contains the first ten residues was able to undergo autoxidation in the presence of ascorbate, contrary to a derivative in which a histidine in the third position was replaced with an alanine.74 This derivative also did not bind copper or nickel, consistent with the view that metal binding is essential for generation of ROS species.74 Histatin 5 is also able to oxidatively cleave DNA76 and catechol with enzyme-like kinetics.77 Hepcidins. Hepcidin-25 is a peptide that plays an important role in iron homeostasis by binding to and regulating the expression of the ferroportin protein.78 The major form of hepcidin is hepcidin-25, but two other forms exist without the Cu-ATCUN motif, hepcidin-22 and hepcidin-20. Hepcidin-25 is able to bind Cu2+, Ni2+, and Zn2+ as determined by UV−vis and mass spectroscopy, and binding of the copper appears to decrease its propensity to aggregate at high concentrations, unlike hepcidin-22 and hepcidin-20.79−81 NMR spectroscopy determined the structure of hepcidin-20 and hepcidin-25 as distorted β-sheets and also suggested that hepcidin-25 could readily aggregate through stacking interactions at the Nterminus.82 In addition to preventing aggregation of the peptide, several studies have demonstrated that the ATCUN motif in hepcidin25 contributes to its activity. An N-terminal derivative of hepcidin-25 was able to cleave plasmid DNA in the presence of reducing and oxidizing agents while a derivative lacking the ATCUN motif showed no cleavage.79 In another study, the antimicrobial activity of hepcidin-25 was increased in the presence of copper, suggesting that the ATCUN motif may play a role in its ability to inhibit bacteria.83 The ATCUN motif may also be of importance for hepcidin’s role in iron homeostasis. Replacing the third histidine with alanine resulted
antibacterial adhesion activity, relative to the histatin derivative with the rigid linker. Lastly, a human β-defensin 3 derivative (HBD3) was also engineered with a triglycine linker and displayed antibiofilm and broad-spectrum activity, albeit at higher than desired concentrations.69 While higher concentrations of these peptides are generally required, the addition of the titanium binding motif may contribute to their low cytotoxicity by effectively immobilizing and orienting the peptide, while also preventing release into solution.
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ANTIMICROBIAL PEPTIDES CONTAINING THE Cu-ATCUN MOTIF The amino terminal Cu2+ and Ni2+ (ATCUN) binding motif is found in several proteins, the most abundant of which are albumins (Figure 7).70 The ATCUN motif requires a free
Figure 7. Structure of the amino terminal copper and nickel binding (ATCUN) motif with the sequence XXH followed by the remainder of the AMP sequence. X is any amino acid except for proline, and M2+ can be Cu2+ or Ni2+.
amino-terminus, a histidine residue at the third position of the peptide or protein, and two deprotonated backbone amide nitrogen atoms to coordinate metal.70 The addition of the ATCUN motif to small peptides has been shown to promote efficient chemistry for numerous biomolecules including angiotensin converting enzyme, sortase A, the HCV internal ribosomal entry site RNA, and the HIV Rev response element RNA.71 In addition to finding the ATCUN motif in proteins, several antimicrobial peptides possess a naturally occurring ATCUN motif in their primary structure (Table 1). Histatins. Histatins are histidine-rich peptides that are secreted by the salivary glands in humans and display both antimicrobial and antifungal activity.72 Histatin 5 and histatin 3 both contain Cu-ATCUN sequences and are able to bind Cu2+ and Ni2+ with high affinity and Zn2+ with a lower affinity as
Table 1. Summary of Naturally Occurring AMPs Containing ATCUN Motifs peptide and sequence
target organisms
histatin 3, DSHAKRHHGYKRKFHEKHHSHRGYRSNYLYDN-COOH histatin 5, DSHAKRHHGYKRKFHEKHHSHRGYCOOH hepcidin 25, DTHFPICIFCCGCCHRSKCGMCCKTCOOH
antifungal: Candida Cryptococcus, neoformans, Saccharomyces, cerevisiae antibacterial: Staphylococcus, Porphyromonas gingivalis antibacterial: E. coli, P. aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophila, Klebsiella pneumoniae, S. aureus, S. epidermis
induces formation of ROS, which inhibits mitochondria in C. albicans; mechanism in bacteria is unclear
ixosin, GLHKVMREVLGYERNSYKKFFLR-NH2
antibacterial: E. coli, S. aureus, P. aeruginosa antifungal: C. albicans antibacterial: E. coli, S. aureus, P. aeruginosa
ATCUN motif oxidizes membrane bilayer; promotes synergy with ixosin B
piscidin 1, FFHHIFRGIVHVGKTIHRLVTG-NH2 piscidin 3, FIHHIFRGIVHAGRSIGRFLTG-NH2
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proposed mechanism
regulates iron homeostasis by binding ferroportin protein, which induces internalization of ferroportin; mutational studies suggest ATCUN motif may be critical for hepcidin’s role in iron homeostasis
DOI: 10.1021/acschembio.7b00989 ACS Chem. Biol. 2018, 13, 844−853
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ACS Chemical Biology Table 2. Summary of ATCUN Derivatives of AMPs peptide and ATCUN AMP sequence sh-buforin: DAHRAGLQFPVGRVHRLLRK-NH2, GGHRAGLQFPVGRVHRLLRK-NH2, VIHKRAGLQFPVGRVHRLLRKNH2 WRWYCR: GGHWRWYCR-NH2, GGHWRWYCRGGK-NH2 anoplin: DAHGLLKRIKTLL-NH2, GGHGLLKRIKTLL-NH2, VIHGLLKRIKTLL-NH2 PAP: DAHKLAKLAKKLAKLAK-NH2, GGHKLAKLAKKLAKLAK-NH2, VIHKLAKLAKKLAKLAK-NH2
target organisms
proposed mechanism
E. coli, Enterobacter aerogenes, B. subtilis, S. epidermis
internalizes inside the cell and damages nucleic acid via production of ROS
E. coli, P. aeruginosa, A. baumannii, S. enterica, MRSA, E. faecium E. coli, E. aerogenes, B. subtilis, S. epidermis E. coli, E. aerogenes, B. subtilis, S. epidermis
broad-spectrum nuclease activity generates ROS, which oxidatively damages membrane bilayer oxidizes membrane bilayer through production of ROS
the ATCUN-peptide displays broad-spectrum nuclease activity.96 Anoplin and PAP are two antimicrobial peptides that exhibit broad-spectrum activity toward bacteria by targeting the cell membrane.97−101 ATCUN derivatives of these two peptides also target the cell membrane but have increased antimicrobial activity due to the generation of ROS and oxidation of lipids in the membrane bilayer.94,102 For the ATCUN derivatives of anoplin, the rate of ROS generation correlates with the extent of lipid oxidation.102
in decreased activity in murine models as well as increased iron in the serum.81 Ixosin. Ixosin is secreted in the salivary glands of the hard tick, Ixodes sinesis, and displays activity against bacteria and fungi.84 Its sequence also contains an ATCUN motif (GLH) that is necessary to generate ROS but is not critical for the potency of the peptide.85 Ixosin promotes lipid oxidation of the cell membrane, which increases the affinity of ixosin B for the cell membrane.85 Importantly, this study was the first to demonstrate synergism between two antimicrobial peptides. Piscidin. Piscidins are found in the mast cells of fish and display broad-spectrum activity against bacteria.86,87 Originally, piscidins were believed to exert their activity by forming pores in the membrane,88 but more recent studies have demonstrated that both piscidin 1 and piscidin 3 can localize on the cell membrane as well as intracellularly, with piscidin 3 targeting DNA over the cell membrane.89 The ATCUN motif also plays an important role in the potency of piscidins. Both piscidins oxidize the membrane bilayer and induce damage to the genomic DNA, with piscidin 3 targeting DNA at a faster rate compared to piscidin 1.90 In addition to targeting planktonic DNA, these peptides were also able to inhibit biofilms by cleaving the extracellular DNA.90 This study demonstrates that AMPs containing the ATCUN motif can have complementary effects with other AMPs and also have the potential to target a multitude of microbes including biofilms. Other Naturally Occurring Cu-ATCUN AMPs. In addition to histatin, hepcidins, and ixosin, other AMPs contain ATCUN motifs in their sequence, but no studies have been conducted to elucidate the role of the ATCUN motif. For example, clavanin C showed up to 5-fold increased activity compared to clavanin A, which has a glutamine residue instead of histidine in the third position of the peptide.33 Furthermore, the AMP database (http://aps.unmc.edu/AP/main.php) contains over 40 entries for naturally occurring peptides that contain an ATCUN motif. More work is needed to directly investigate the role of the ATCUN motif in order to clarify whether it contributes to the biological activity of the peptides. Synthetic Cu-ATUCN AMPs. Many studies have evaluated the effect of attaching the ATCUN motif to antimicrobial peptides (Figure 7, Table 2). Buforin II is a peptide that has high homology to the N-terminus of histone H2A and exerts its activity by localizing in the cytosol of the cell and targeting nucleic acids.91−93 Addition of the ATCUN motif to sh-buforin, which lacks the first four residues of buforin II, results in increased ROS production in the cell and decreased MICs.94,95 The ATCUN derivatives of buforin did not permeabilize the membrane, but did oxidatively cleave DNA, and so their target appears to be the same as that of the parent peptide.94,95 The ATCUN motif has also been observed to increase the antimicrobial activity of WRWYCR in a similar manner, since
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CONCLUDING REMARKS AMPs are able to target a broad range of pathogens through multiple mechanisms that include cell membrane disruption or targeting of DNA and RNA. Some AMPs are also able to utilize metals to improve their activity and inhibit pathogens. These AMPs use metals to alter their structure, perform redox chemistry on targets, and sequester essential metals from bacteria. It would not be surprising if more antimicrobial peptides were identified with metal-binding capabilities, especially since thousands of these peptides have already been identified. As a result of increasing antibiotic resistance, it is vital to develop novel therapies that can target resistant pathogens. AMPs are a promising class of molecules due to their broad spectrum of activity toward a multitude of pathogens. It is important to fully understand their mechanism of action, including the key roles served by metal cofactors, as a prelude to the application or design of novel families of metalloantibiotics based on naturally occurring antimicrobial peptides.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
J. A. Cowan: 0000-0002-4686-6825 Author Contributions †
J.L.A. and Z.T. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health (HL093446). REFERENCES
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DOI: 10.1021/acschembio.7b00989 ACS Chem. Biol. 2018, 13, 844−853
