ABSTRACT. Keywords: antimicrobial peptide, peptide-membrane interactions, peptide structure, antimicrobial activity

Transcription

1 Antimicrobial Peptides: A Review of How Peptide Structure Impacts Antimicrobial Activity Jason W. Soares, and Charlene M. Mello* U.S. Army RDECOM, Kansas St., Natick, MA USA * charlene.mello@us.army.mil ABSTRACT Antimicrobial peptides (AMPs) have been discovered in insects, mammals, reptiles, and plants to protect against microbial infection. Many of these peptides have been isolated and studied exhaustively to decipher the molecular mechanisms that impart protection against infectious bacteria, fungi, and viruses. Unfortunately, the molecular mechanisms are still being debated within the scientific community but valuable clues have been obtained through structure/function relationship studies 1. Biophysical studies have revealed that cecropins, isolated from insects and pigs, exhibit random structure in solution but undergo a conformational change to an amphipathic α-helix upon interaction with a membrane surface 2. The lack of secondary structure in solution results in an extremely durable peptide able to survive exposure to high temperatures, organic solvents and incorporation into fibers and films without compromising antibacterial activity. Studies to better understand the antimicrobial action of cecropins and other AMPs have provided insight into the importance of peptide sequence and structure in antimicrobial activities. Therefore, enhancing our knowledge of how peptide structure imparts function may result in customized peptide sequences tailored for specific applications such as targeted cell delivery systems, novel antibiotics and food preservation additives. This review will summarize the current state of knowledge with respect to cell binding and antimicrobial activity of AMPs focusing primarily upon cecropins. Keywords: antimicrobial peptide, peptide-membrane interactions, peptide structure, antimicrobial activity 1.0 BACKGROUND Insects, mammals, reptiles, and plants all produce antimicrobial peptides (AMPs) to protect against microbial infection and ensure survival in ever-changing environments. Hundreds of antimicrobial peptides have been discovered and classified based upon their structure/activity relationships 3. These studies have enabled the majority of the peptides to be classified into two groups. 3,4 Peptides with β-sheet conformation and one or more disulfide bonds represent one classification which includes defensins isolated from mammals 5,6,7 and insects. 8,9 This paper will focus upon the second class of AMPs pertaining to the linear, cationic peptides which are generally random-coil in solution but form amphipathic helical structures upon interaction with a cell membrane. Examples include melittin isolated from honeybee venom 10, cecropins isolated from insects 11 and mammals 12, and magainins isolated from Xenopus laevis frog skin. 13,14 Antimicrobial peptides have a diverse and broad spectrum of antimicrobial activity towards gram-negative and gram-positive bacteria (see TABLE I), fungi 15, and viruses 16,17. Many AMPs, including both insect and mammalian cecropins, are unique due to their lack of hemolytic activity while other AMPs, such as melittin and magainins (at high concentrations), were found to be highly toxic in mammals. 10,13 Cecropins are not lethal to Chang liver cells 18, sheep red blood cells 19, human erythrocytes, 20 or normal lymphocytes 21 but will selectively kill bacterial cells. In addition, cecropins display the ability to lyse leukemia cancer cells 22,21 and have shown potential as a drug delivery system, masking the toxic effects of current anti-cancer drugs while still delivering them to a cancer cell. 21,23 A major challenge in the analysis of antimicrobial peptides is the diversity of mechanisms of interaction between peptides and cellular membranes 2. The majority of the analyses have been focused on insect cecropins isolated from the Hyalophora cecropia moth 11 and the mammalian cecropin isolated from pig intestine 12. Insect and mammalian cecropins (TABLE II) are structurally 24 similar both being linear, cationic peptides which exhibit random coil structure in solution and form amphipathic α-helical structures upon interaction with a cellular surface. 25 We will discuss investigations focused on insect cecropins, specifically Cecropin A, B, D and analogues of all, and explore the effect of modifying sequence, charge, and hydrophobicity has on activity. 20 Monitoring Food Safety, Agriculture, and Plant Health, edited by Bent S. Bennedsen, Yud-Ren Chen, George E. Meyer, Andre G. Senecal, Shu-I Tu, Proceedings of SPIE Vol (SPIE, Bellingham, WA, 2004) X/04/$15 doi: /

2 2.0 STRUCTURE/FUNCTION RELATIONSHIPS 2.1 Mechanisms of Interaction The molecular mechanisms of antimicrobial peptides are an ongoing topic of debate but several research teams have obtained great insight into these mechanisms. Insect and mammalian cecropins have been the most extensively studied peptides to elucidate the properties important for binding to bacterial cells. Christensen et al. determined that cecropins form voltage-dependent, time-variant ion channels in planar lipid membranes. 26 These pores or channels were a result of peptide binding and insertion into the lipid bilayer leading to cell lysis. Not surprisingly, they also demonstrated that cell lysis is dependent on peptide length; peptide fragments were inserted into the lipid bilayer but cell lysis did not occur presumably due to their inability to span the bilayer. Insect cecropins were studied by Steiner et al. correlated peptide binding and membrane activity in a concentration-dependent mechanism through the formation of a peptide monolayer on the bacterial membrane surface causing gradual destabilization of the membrane and cell lysis. Interestingly, some bacteria not susceptible to cecropin were still found to bind the peptide without subsequent cell lysis. 19 This suggests that a peptide may have multiple modes of interaction with a specific bacterial species and may acquire binding selectivity. Marassi et al. asserts that cecropin A binds electrostatically and parallel to the lipid membrane, self-aggregates to insert across the lipid membrane and forms pores when the peptide concentration has reached a critical level. 27 Wade et al. further investigated membrane insertion by designing D and L enantiomers of cecropin A. Both enantiomers produced equivalent single-channel conductance; suggesting that the mechanism is not dependent upon the chiral nature of the peptide. 28 In subsequent studies, Silvestro et al. analyzed cecropin A folding into its fully active structure by looking at intermediate stages of monolayer absorption. 29 Cecropin A was found to fold into primarily α-helical secondary structure and initially orient parallel to the membrane surface. Cecropin B discovered in Heliothis virescens larvae has been implicated in a proposed pore formation mechanism. 30 This may be an indication that cecropins isolated from different sources may interact similarly with bacterial membranes. Chan et al. produced SEM micrographs of cecropin B and structural analogues of this peptide to reveal bleb-like protrusions on the cellular membrane surface depicting cellular damage. 22 Further investigation with TEM elucidated swelling and shrinking phenomena of the cells as a result of the peptide interaction within the membrane and the passage of water into and out of the cellular matrix through pores formed by the peptide. Changes in the dimension and shape of the cell were also reported. These studies support previous predictions of pore formation in bacterial membrane systems. Mammalian cecropin P1 has been reported to have a non-pore forming mechanism within phospholipid membranes. This carpet-like mechanism has been proposed as an initial interaction with the phospholipid membrane leading to an accumulation of peptide monomer on the cellular surface, re-orientation, and subsequent cell lysis occurring through disintegration of the lipid membrane. 31 This is similar to the monolayer accumulation non-pore mechanism proposed by Steiner et al. for insect cecropins. 19 Haukland et al. used TEM to investigate mechanism similarities between magainin and cecropin P1 and found magainin migrated through the cytoplasmic membrane into the cytoplasm while cecropin P1 resided exclusively in the cell wall. 32 In contrast to all of the cecropin research, a linear cationic peptide, buforin II from stomach tissue of the Asian toad Bufo bufo garagriazan 33, was found to penetrate the bacterial membrane without displaying membrane permeabilization and accumulate in the cytoplasm targeting nucleic acids. 34 These studies determined that the proline hinge of buforin II is essential for activity since removal of the hinge resulted in an accumulation of the peptide on the cell surface. The importance of understanding the biophysical properties that drive these peptide-cell interactions and subsequent antimicrobial activity is essential to rationally designing peptides with better specificity and selectivity. 2.2 Amphipathic α-helical structure Merrifield et al. was able to show that the N-terminal region of cecropin A formed an amphipathic α-helical structure upon contact with a hydrophobic membrane. A disruption of the helical structure occurs at the Gly-Pro residues centrally located in the sequence. 35 This helix-hinge-helix conformation is indicative of insect cecropins (see Table II) but mammalian cecropins lack this hinge structure and consist of an amphipathic α-helix spanning the entire molecule. 12 Slight differences in sequence affects the potential mechanism of action that in turn affects the antimicrobial activity of each peptide. 31 Lee et al. investigated the specific sequence homology between cecropin P1, cecropin A, and Proc. of SPIE Vol

3 magainin. 12 Cecropin P1, similar to magainins, displayed a free carboxyl group at the c-terminus while the insect cecropins are amidated. Synthetic forms of cecropin P1 revealed that upon amidation of the c-terminus, the antimicrobial activity towards gram-positive bacteria increased. Chan et al. used TEM to investigate the actual separation between the inner and outer membranes of the gramnegative bacteria Klebsiella pneumonia by induction with cecropin B. The structure of cecropin B comprises an N- terminal amphipathic helix separated from a hydrophobic helix by the characteristic Gly-Pro hinge. A swelling phenomenon was observed by the natural cecropin B and structural analogues of this peptide were the c-terminal hydrophobic helix was replaced by an amphipathic α-helix. Furthermore, these analogues resulted in only a slight reduction of antimicrobial activity. 22 However, when the N-terminal amphipathic helix was replaced with a hydrophobic helix no antimicrobial activity was observed confirming the necessity of an amphipathic structure for antimicrobial activity. Andreu et al. created a series of N-terminal analogues of cecropin A substituting amino acids with helix promoting, helix forming, and helix breaking amino acid residues. 36 Circular dichroism was used to examine the affect these substitutions had on secondary structure formation by solvent induction. Although it was difficult to correlate the degree of helicity with antimicrobial activity, single amino acid replacements, particularly the tryptophan residue in position 2, significantly decreased antimicrobial activity. Magainin II and a magainin II-amide were investigated by Abler et al. Both were tested to determine their affects upon known food pathogens under a range of conditions including temperature and protein constituents. The magainin II had a broad inhibitory activity although, after an extended period of time, the organisms recovered. The magainin II amide completely inactivated the organisms and enhanced antimicrobial activity at higher temperatures. 37 These investigators proposed the amide form of the peptide was likely to form more stable α-helices than the natural magainin thus enhancing the bioactivity. 2.3 Amino acid chirality, sequence and charge Vunnam et al. investigated the importance of the structure and chirality of the cecropin P1 sequence with a more in-depth analysis of D and L enantiomers, free and acetylated N-terminus, amidated and free carboxyl group at the C-terminus, and retro isomers designed by synthesizing the reverse (C to N) sequence. 38 As previously noted, the amidated cecropin P1 was shown to have slightly greater activity against gram-positive bacteria while the acetylation of the N-terminus completely inactivated cecropin P1. The naturally free amino terminus was active as expected. The retro isomers of cecropin P1 were much less active than the parent peptide and possessed hemolytic activity in sheep red blood cells, which the parent peptide did not. No dramatic differences were observed between the D and L enantiomers suggesting chirality does not affect activity but sequence and amidation are the most significant factors. Similar studies were conducted with a proline-arginine rich peptide PR-39, isolated from pig intestine. 39 In this case, chirality played a much more significant role in activity. The L isomers were inactive against Staphylococcus aureus but the D isomers showed remarkable activity versus the same organism. 38 Natural peptide sequence, helical dipole direction, and free amino terminus of cecropin P1 are essential to the peptide behavior and modifying these characteristics significantly affect antimicrobial activity, but when analyzing a different peptide, the same type of changes did not yield similar results. In fact, the results of modifying the PR-39 peptide suggest that peptide selectivity and antimicrobial activity are impacted by chirality. Hong et al. synthesized a decapeptide and investigated the effect of charge upon its activity. 15 The researchers determined that a lower ph correlated to an increase in net positive charge enhancing antifungal properties of the designed peptide while retaining the antimicrobial activity towards gram-positive bacteria. Appendini et al. recently demonstrated a synthetic antimicrobial peptide containing six leucine and eight lysine residues having the ability to kill food microorganisms including yeast although the activity was affected by changes in ph. 40 Cell death of the food pathogen, Escherichia coli O157:H7, was significantly increased in more acidic conditions. 2.4 Role and identification of essential amino acids Many investigators have proposed that the entire native peptide sequence is not essential for bactericidal activity or binding. It has also been suggested that several different binding domains within a single peptide may be responsible for the selectivity observed. Investigation of synthesized N-terminal analogues of Cecropin A confirmed the importance of the tryptophan residue in position 2 for retention of antimicrobial activity 36. In addition, Kragol et al. identified crucial amino acid residues in the proline-rich peptide, pyrrhocoricin. 41 Modification of the N-terminus through alanine replacements demonstrated that amino acids 2-10 are crucial residues responsible for antimicrobial 22 Proc. of SPIE Vol. 5271

4 activity. Furthermore, the C-terminus was identified as essential for delivery of the peptide into the cell. These results suggest that cell lysis and cell penetration are the result of two different regions of the peptide sequence, which may be beneficial as potential uses for drug delivery by customizing the design of shorter peptides. Finally, a series of synthetic arginine and tryptophan-rich peptides were designed by Strom and co-workers based upon the role these amino acids play in cell death 42. The research determined a minimum of three arginine and three tryptophan residues were required for antimicrobial activity without the need for a specific order of the residues within the peptide. 2.5 Peptide Development A better understanding of the structure/function relationships, identification of binding domains, and essential amino acids for enhancement of antimicrobial activity has lead to the design of peptide hybrids. 3 Park et al. synthesized several hybrid peptides blending the leucine (L)-lysine (K)-rich amphipathic portion of cecropin A and the hydrophobic region of magainin II 20. Enhanced antimicrobial, antitumoral, and antifungal affects were observed as well as the elimination of hemolytic activity that is present in native magainin II. Boman et al. synthesized a series of cecropin A- melittin hybrids identifying a hybrid that exhibited markedly improved antimicrobial activity and antimalarial properties. This hybrid peptide has the ability to overcome the resistance of some gram-positive species as well as masking the hemolytic activity of melittin. 43,28 Friedrich et al. developed a new series of cecropin-melittin hybrids and analyzed the hybrids for activity and salt resistance. 44 Researchers demonstrated the ability to effectively maintain antimicrobial activity in an elevated salt environment just through changes in hydrophobicity, charge, and amphipathicity. This hybrid analog displayed the ability to be salt-resistant in the presence of NaCl although activity reduced significantly in much less MgCl CONCLUSION The mechanism of cellular interaction is still being debated but it has been accepted that cecropins and magainins behave in a carpet like manner 31,19 either forming pores or causing complete membrane disintegration leading to cell death while others peptides, buforin II in particular, may penetrate the cell membrane effecting protein or DNA synthesis. Specific amino acids have a large impact upon the mechanism as it has been suggested with the buforin II proline hinge 34 and the proline-glycine hinge which separates the two amphipathic helical structures of the insect cecropins 35. Analyzing all the research on peptide-cell mechanisms has elucidated the possibility that each antimicrobial peptide may have a unique mechanism of interaction with a cell. The affects of salt, temperature, protein constituents, and ph must still be further investigated but the opportunities to tailor peptides and maintain activity in these environments are authentic. The elucidation of binding domains and essential amino acid motifs are giving researchers the ability to create new peptides possessing enhanced selectivity and potency. These new peptides will are also playing a key role in the investigation of cell binding mechanisms. The ability to penetrate cell membranes creates the potential for antimicrobial peptides to be used as drug delivery agents. In addition membrane-acting peptides, such as cecropin, have the ability to be used as antibiotics with their broad spectrum of activity versus bacterial and cancer cells. 45 Current antibiotics, especially those for treatment of cancer, show cytotoxicity against human cells while some antimicrobial peptides do not; providing an opportunity for peptides to mask the toxicity of anticancer drugs. 21 These studies also suggest that tailored AMPs may provide safe alternative to traditional antibiotics; potentially overcoming bacterial resistance. ACKNOWLEDGEMENTS We thank Dr. Andre Senecal for the ongoing support of the peptide program at the RDECOM in Natick, Massachusetts. Also would like to thank Mr. Steven Arcidiacono for in-depth discussion and direction as well as Ms. Kimberly Morin for her continued technical assistance. Proc. of SPIE Vol

5 Gram-negative Gram-positive Antimicrobial Peptide E. coli D21 P. aeruginosa B.subtilis S. aureus Cecropin A a,b >300 Cecropin B b Cecropin P1 c >520 Magainin I a Melittin a CA(1-8)-MA(1-12) d Not tested Not tested CA(1-8)-M(1-18) a,e Table I. Antimicrobial Activity (MIC:µM) of select peptides towards gram-negative and gram-positive bacteria. Cecropins show a broad range of antimicrobial activity showing slight selectivity of gram-negative versus gram-positive bacteria. Melittin and magainin show a broader range of activity relative to cecropins but display cytotoxicity in mammalian cells. CA-MA hybrid also reveals good activity (12µM) towards Candida albicans. Cecropins and magainins also have potent activity against common gram-negative food pathogens such as E. coli O157:H7 and S. typhimurium (data not shown). a MIC taken from REF 28 b MIC values taken from REF 24,23 c MIC values taken from REF 12 d MIC values taken from REF 20 e MIC values taken from REF 3,44 24 Proc. of SPIE Vol. 5271

6 Peptide Source Sequence Cecropin A a Hyalophora cecropia, moth kwlfkkiekvgqnirdgiikagpavavvgqatqiak Cecropin B a Hyalophora cecropia, moth kwkvfkkiekmgrnirngivkagpaiavlgeakal Cecropin D a Hyalophora cecropia, moth wnpfkelekvgqrvrdavisagpavatvaqatalak Cecropin P1 a Porcine small intestine swslsktakklensakkrisegiaiaiqggpr PR-39 b Porcine small intestine rrrprppylprprpppffpprlpprippgfpprfpprfp Cecropin A c Aedes albopictus, vector mosquito gglkklgkklegvgkkrvfkasekalpvavgikalg Magainin I d Xenopus laevis, frog skin gigkflhsagkfgkafvgeimks Magainin II d Xenopus laevis, frog skin gigkflhsakkfgkafvgeimns Buforin I e Bufo bufo gargarizans, Asian toad agrgkqggkvrakaktrssraglqfpvgrvhrllrkgny Buforin II e Bufo bufo gargarizans, Asian toad trssraglqfpvgrvhrllrk Defensin, NP1 f Rabbit neutrophils vvcacrralclprerragfcrirgrihplccrr Insect Defensin A g Phormia terranovae, dipteran atcdllsgtginhsacaahcllrgnrggycngkgvcvcrn Melittin h Honey bee venom gigavlkvlttglpaliswikrkrqq Table II. Short list of Antimicrobial peptides of potential interest. The Gly-Pro hinge is characteristic of the insect cecropins helix-hinge-helix conformation (presented in bold text). The mammalian cecropin P1 display an extended α- helix throughout the molecule due to presence of this Gly-Pro motif at the c-terminus. Both insect and mammalian defensins contain multiple cysteine residues that are not present in any of the cecropins. a Sequence and source from Ref 24 b Sequence and source from Ref 39 c Sequence and source from Ref 46 d Sequence and source from Ref 14 e Sequence and source from Ref 33 f Sequence and source from Ref 7 g Sequence and source from Ref 8,9 Sequence and Source from Ref 10 Proc. of SPIE Vol

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