Antimicrobial Peptides

Abstract

Background:

Bacterial resistance to available antibiotics has resulted in enhanced efforts at antibiotic stewardship but also has led to investigation into alternative methods for managing surgical infections. Antimicrobial peptides (AMPs) are naturally occurring compounds produced by all prokaryotic and eukaryotic cells that have potential as an alternative to conventional antibiotics.

Methods:

The published literature was reviewed for investigations that were relevant to infections commonly seen by surgeons and the potential applicability of AMPs for surgical care.

Results:

Antimicrobial peptides are low-molecular-weight peptides with activity against bacteria, fungi, and viruses. Experimental evidence shows that AMPs have activity against highly resistant bacteria identified from human infections. Furthermore, these peptides can be designed as semi-synthetic or totally synthetic constructs for potential clinical use. Antimicrobial peptides appear to have in vivo activity in limited animal studies, but the experimental models for evaluation of these peptides need more clinical relevance. These products are in clinical evaluation at present but are limited in number and are being evaluated primarily for topical applications.

Conclusions:

Antimicrobial peptides have considerable in vitro evidence that supports their use for the prevention and treatment of surgical infections. Better experimental and clinical trial efforts are needed to move this technology toward applicability in surgical care.

Antimicrobial peptides (AMPs), also known as host defense peptides, are a vast array of naturally occurring compounds that are best known for a broad scope of antimicrobial activity against bacteria, virus, fungi, and protozoa. These compounds are amino acid peptides, which differentiates them from conventional antibiotics. Conventional antibiotics (e.g., penicillins), most of which are naturally occurring compounds that have components of carbon, nitrogen, sulfa, and other chemical elements but not amino acids. Exceptions to the absence of amino acids in current antibiotics are glycopeptides (e.g., vancomycin) or lipoproteins (e.g., daptomycin). Conventional antibiotics generally are identified with specific activity against only bacteria. Like conventional antibiotics, each AMP has a specific microbial target, but the aggregate spectrum of different AMPs covers viruses and fungi as well as most bacterial species. Furthermore, AMPs can be produced by plants, bacteria, fungi, and eukaryotic cell populations, which can provide a large number of prototype peptides for study and biochemical manipulation.

Both AMPs and penicillin were independently recognized in the 1920s by Alexander Fleming. In 1922, he identified lysozyme as the prototypical AMP by identification of the in vitro antibacterial effects of human nasal discharge [1]. Penicillin was recognized some seven years later [2], and it was penicillin that was carried forward for clinical application and commercial production. Early therapeutic efforts with AMPs failed because of toxicity considerations and difficulties/costs in production, and for that reason, these compounds were put aside, and conventional antibiotics were pursued for clinical application. However, at present, the progressive emergence of gram-positive and gram-negative bacterial organisms resistant to conventional antibiotics has resulted in renewed interest in AMPs as potential treatments. Little information is available in the surgical literature about the potential applications of AMPs for prevention or therapy of surgical infections, but extensive scientific investigation in the microbiology and biochemistry literature has documented these peptides as having potential applications in clinical medicine. This overview will examine AMPs as potential therapies for the surgical patient. It is in no way a comprehensive presentation on this vast subject. Rather, it is intended to be an introduction for surgeons to the potential of AMPs as a source of antimicrobial therapy for the future and the need for surgical engagement in this class of potential treatments [2].

Molecular Structure and Mechanisms of Action

Antimicrobial peptides are diverse in structure with variability that is programmed by the cell source of the peptide. More than 2,000 unique naturally occurring AMPs have been identified, and more than 100 human AMPs are currently known. This proliferation of unique peptides has resulted in multiple AMP databases from different parts of the world that have catalogued a wide array of these molecules from different sources [3 –8]. With the potential for biochemical manipulation for therapeutic targets, the number of semi-synthetic and synthetic AMPs is likely to expand considerably in coming years.

Antimicrobial peptides have from five to about 50 amino acids and have secondary α-helix, β-sheets, and other configurations [9,10]. These secondary structures provide the functional specificity of each peptide. They are amphipathic molecules with a hydrophilic and hydrophobic end. Of interest, some AMP molecules demonstrate no secondary structure in an aqueous environment but assume a secondary structure when exposed to lipid such as the bacterial cell membrane. These peptides are produced constitutively but may be induced when active infection or bacterial endotoxin is sensed by host cells. The programming for most AMPs is part of a larger protein, with the biologically active AMP being cleaved from the parent molecule [11].

The majority of AMPs are cations and therefore bind electrostatically to the anionic targets of specific microbes. The magnitude of the positive charge differs by AMP, and this net charge is of considerable significance in the affinity with which the molecule binds to the anionic target site. The anionic target for gram-negative bacteria is the lipopolysaccharide of the outer membrane. Injury to the outer membrane by binding of the AMP results in penetration and depolarization and loss of ionic segregation of important intracellular solutes. Pores may be created in the cell membrane, which causes extravasation of intracellular proteins and ultimately leads to lysis of the bacterium. This target specificity of AMPs for lipopolysaccharide has been proposed for use in anti-endotoxin treatment for patients with gram-negative sepsis [12].

Antimicrobial peptides that are specific for gram-positive bacteria have an anionic target of the peptidoglycan or lipoteichoic acid component of the cell wall [13] and the lipid component of the cell membrane. The cell wall components are thought to entrap the AMPs and prevent access to the ultimate plasma membrane target. Thus, loss of cell wall function by saturation of anionic binding sites permits penetration of the AMP to the cell membrane. As opposed to the lipopolysaccharide target of gram-negative bacteria, the principal cell membrane target of AMPs for gram-positive bacteria is anionic phosphatidylglycerol [14]. Staphylococcus aureus has 80% of its membrane lipid as phosphatidylglycerol, but gram-negative bacteria have less than 25% [15]. Differences in the composition of membrane lipids accounts for the specificity of selected AMPs for particular bacterial species.

The specificity of AMPs for the anionic targets of the bacterial cell membrane spares mammalian cells from being affected by the peptides. The human and other mammalian cell membranes contain zwitterions of phospholipids and cholesterol [16], and any negatively charged sites are expressed on the intracellular surface of the lipid membrane and are protected from external cationic AMPs.

A small number of AMPs are anions and have a different mechanism of action than do the cationic peptides. A notable human anionic AMP is dermcidin, which is present in sweat and appears to have a role in the regulation of bacterial colonization of the skin [17]. The anionic AMPs have a similar length of amino acids, similar configuration, and amphipathic characteristics like the cationic peptides [18]. They appear to use metallic ion bridges to facilitate the antimicrobial action on the targeted bacterial plasma membrane. The full mechanism of action remains to be defined, but the existence of the anionic variant of AMPs illustrates the enormous diversity of these peptides.

Whereas AMPs target the microbial plasma membrane, selected peptides pass through the cell membrane and affect intracellular targets [19,20]. Intracellular effects include inhibition of DNA, RNA, and protein synthesis; disruption of other cellular synthetic processes; and possibly apoptosis mechanisms. The antibacterial effects are associated with rapid death of the micro-organism. The majority of investigation has been on bacterial effects, but similar mechanisms of action are understood for the targets of AMPs in viruses [21], fungi [22], and parasites [23].

Antimicrobial peptides are being investigated for their immunomodulatory functions [24]. These peptides appear to promote chemotaxis, release histamine, and dampen the excessive pro-inflammatory response that occurs from endotoxin binding to Toll-like receptors, but they may also serve as an “alarmin” signal secondary to injury or active infection. These peptides appear to both stimulate a proinflammatory cytokine response through Toll-like receptors and modulate the effects of endotoxin at this same receptor site [25]. The AMPs appear to play a role in the adaptive immune response, angiogenesis, and wound healing. The native chemokine signals from human monocytes that are associated with being chemotactic signals appear to have antibacterial properties as well. The biological links between AMPs and pro-inflammatory signaling of the human host are becoming compelling as our understanding of these peptides expands.

Specific Peptides

The number of specific AMPs from micro-organisms, insects, vertebrates, plants, and humans continues to expand. Research into these specific peptides identifies unique features of specific AMPs but also reveals common features that all share. This effort at comparative studies of the different AMPs has the promise of identifying the unique amino acid signatures that offer opportunities for engineering synthetic peptides.

Bacteriocins are the AMPs that are derived from bacteria [26]. Bacteria produce AMPs to eliminate competitive micro-organisms and preserve their environmental milieu. Most bacteria produce bacteriocins, and all have immune proteins to protect against self-destruction by their own AMPs. Gram-negative bacteria produce colicins (high molecular weight) and muricins (low molecular weight) that attack by penetrating the cell membrane and damaging the synthetic processes of the target bacteria. Gram-positive bacteria produce three classes of bacteriocins: Class 1 (<28 amino acids) are called lantibiotics collectively because they contain the non-protein amino acid lanthione; Class 2 (30–60 amino acids) are non-lanthione peptides; and Class 3 are high-molecular-weight products. The specific AMPs of gram-negative and gram-positive bacteria have a broad range of activity against targets on most other bacterial species.

Fungi were the source of the original AMP, penicillin discovered by Fleming, and fungi also produce a number of other AMPs with bacteria as the principal target. Plecsin [27], eurocin [28], and copsin [29] are notable as fungal AMPs that target the cell membrane enzyme lipid II of bacteria. This results in inhibition of peptidoglycan synthesis analogous to that seen with vancomycin. Of interest, copsin has activity against vancomycin-resistant Enterococcus faecium. Generally, the fungal AMPs have activity against gram-positive bacteria.

A vast array of AMPs has been identified in plants [30]. These peptides have very similar mechanisms of action targeting bacteria and fungal pathogens that also are observed among AMPs from other microbial or animal sources. Although the mechanisms of cell injury are similar, the composition of the membrane target can be different. Plant AMPs target sphingolipids in the fungal cell membrane but may have antimicrobial activity against both bacteria and fungi [31]. The unique features of plant AMP interactions with potential pathogens underscores the need to understand the primary and secondary structure of the peptide for future focused synthetic constructs.

Insect AMPs have been identified in large numbers and have diverse biological activity. Insects are vulnerable to infection from bacteria, fungi, viruses, and protozoans, so it is logical that unique AMPs have been identified against all of these targets [32]. Cecropin was the first insect AMP identified, from moths [33]. Cecropins are differentiated from other AMPs by virtue of common sequences of 31–39 amino acids in the peptide chain. There are biochemical differences among the various cecropin AMPs, with each specific variant having a unique spectrum of microbial activity. Some cecropins kill bacteria, others inhibit bacterial growth, and still others induce fungal apoptosis [34].

Defensins are the most extensively studied of the insect AMPs. This group of peptides has a signature chemical structure (six conserved cysteine residues with disulfide bridges). Although commonly studied in insects, defensins appear to be present throughout all living cells. Insect defensins have longer (34–51) amino acid chains than cecropins and have activity against gram-positive organisms principally. The variability of activity of the cecropins and defensins as a function of unique amino acid and secondary peptide structures might inform a better understanding of AMP biochemistry.

Virtually all species of the animal kingdom produce AMPS. Invertebrates [35], amphibians [36], shrimp [37], cattle [38], and many others have been found to have AMPs that are adapted for each species-specific need for protection from the microbial world. Regardless of the species source, the common feature of all AMPs is the relatively short peptide chain, the constant cell-membrane binding and injury of the target microbe, and the variability in the specific microbial target.

At the current time, there are 125 human AMPs [39]. Of these, human defensins and cathelicidins have been most intensively studied [40]. Human defensins share the characteristic six cysteine residues with bridging disulfide bonds of defensins found in other species. There are six unique α-defensins and 31 β-defensins [41]. There are few amino acid sequence differences among the various human defensins, but these modest changes impact the activity of the individual peptides. In addition to antibacterial effects, the defensins are active against many pathogenic human viruses and have viral protein and glycoproteins as specific targets. Cathelicidins are structurally different from defensins, but they share the same anti-bacterial, anti-fungal, and anti-viral characteristics [42]. Both cathelicidins and defensins have an array of immunomodulatory functions that make them important parts of the innate immune response in man. The chemical classification and functional specifics of the many human AMPs are discussed in detail elsewhere [43].

Potential Clinical Applications of AMPs

As the 100^th anniversary of Fleming's description of AMPs approaches, it is of interest that there has been limited clinical use of these peptides [44]. They have the positive features of being small molecular weight, an appropriate scope of anti-microbial activity, and minimal antigenicity for the host. Because the cellular target for AMPs is the intrinsic anionic character of the bacterial cell membrane, the likelihood of resistance emerging should be less of an issue than is observed with conventional antibiotics. The successful but limited development of AMPs (Table 1) for the treatment of bacterial infection indicates that the promise is there for the development of novel therapies in upcoming years [45 –53].

Table 1.

Antimicrobial Peptides Being Used for Treatment of Bacterial Infections in Clinical Practice

Product	Infection target	Comment
Bacitracin [45]	Gram-positive bacteria	Commonly used topical cyclic peptide derived from B. subtilis; too toxic for systemic use
Vancomycin [46]	Gram-positive bacteria	Glycopeptide; derived from Amycolatopsis orientalis, most commonly used for methicillin-resistant Staphylococcus aureus infection
Daptomycin [47]	Gram-positive bacteria	Lipopeptide; 13 amino acids, derived from Streptomyces roseosporus, used principally for bacteremia and soft-tissue infections caused by gram-positive organisms
Teicoplanin [48]	Gram-positive bacteria	Complex semi-synthetic glycopeptides; derived from Actinoplanes teichomyceticus; inhibits cell wall synthesis. Not used in the U.S.
Dalbavancin [49]	Gram-positive bacteria	Semi-synthetic lipoglycopeptide; derived from Nonomuria spp.; once-weekly administration; inhibits cell wall synthesis
Oritavancin [50]	Gram-positive bacteria	Semi-synthetic lipoglycopeptide; derived from chloroeremomycin from Amycolatopsis orientalis, 200–300 h half-life
Telavancin [51]	Gram-positive bacteria	Lipoglycopeptide derivative of vancomycin; 9 h half-life
Polymyxin B [52]	Gram-negative bacteria	Derived from Bacillus polymyxa; used as a topical ointment for soft tissue wounds; used systemically for resistant gram-negative infections
Colistin [53]	Gram-negative bacteria	Cyclic polypeptide derived from Bacillus polymyxa; used for resistant gram-negative infections

Surgical site/soft tissue infections

Glycopeptides and lipopeptides have already been used for the prevention and treatment of surgical site infections (SSIs) and have been used extensively in complicated skin/skin structure infections [54,55]. In vitro evidence of numerous naturally occurring AMPs effective against methicillin-resistant S. aureus (MRSA), enterococcal pathogens, and resistant gram-negative isolates suggests that the potential exists for prevention and treatment of these serious organisms that are common in SSIs and other soft tissue infections [56]. Björn et al. [57] demonstrated significant killing of Candida albicans and MRSA in experimentally contaminated mouse soft tissue wounds with a lactoferrin-derived topical AMP. These same authors demonstrated experimental benefit with another AMP in the treatment of Pseudomonas aeruginosa burn infection in mice and in SSI [58,59]. Chalekson et al. [60,61] studied the AMP D2A21 and demonstrated a significantly better survival rate than in control animals in experimental burns and open wounds infected with Pseudomonas aeruginosa. Prosthetic devices and titanium implants have been coated with AMPs [62]. Even wound dressings employing vitamin D with nanofiber technology are being designed to optimize the endogenous release of AMPs to prevent SSIs [63]. Investigation into the prevention and management of SSI and soft tissue infections requires clinically relevant experimental infections to provide direction for the use of AMPs [64].

Biofilm inhibition

A biofilm is a community of bacteria attached to a biological or artificial surface that is encased in an extracellular matrix of polysaccharides, proteins, lipids, and nucleotides. Bacteria in an extracellular matrix are functionally in a “safe haven” where immune elements and antibiotics are unable to access them. Sub-therapeutic antibiotic penetration into the biofilm leads to progressive microbial resistance. These challenges are especially difficult for surgical infections on implanted prosthetic materials (e.g., total hip prostheses) and on indwelling catheters and other devices used in patient management (e.g., urinary tract catheters) [65]. Antimicrobial peptides have a potential role in disrupting the ecology of biofilms and the extracellular matrix. Naturally occurring and synthetically constructed AMPs have demonstrated in vitro promise in managing these infections. Cationic AMPs, as smaller molecules, can penetrate the extracellular matrix, and the natural anionic target on the cell membrane of the bacteria makes this an area of research focus [66 –68].

Urinary tract infections

Urinary tract infection in the catheterized and elderly surgical patient continues to be a major source of complications. Preventive strategies have been limited, and resistant pathogens for these hospital-acquired infections are problematic. An interesting relation between urinary tract AMPs [69], vitamin D [70], and microorganisms that naturally reside in the urinary tract and those that are introduced by healthcare instrumentation is evolving [71].

Antimicrobial peptides are proposed for both prevention and treatment of these infections [72]. The role of biofilms on the surface of urinary catheters is yet another part of this complex conundrum [73]. However, early experimental evidence of urinary catheter infections in mice indicates a potential role for AMPs [74].

Pneumonia

Post-operative hospital-acquired and ventilator-associated pneumonias continue to be major complications among critically ill and elderly surgical patients. This is a particularly interesting area for application of AMPs, as daptomycin as a lipoprotein antimicrobial agent has proved to be ineffective for pneumonia because of its poor tissue penetration [75]. Hou et al. [76] demonstrated that both natural and synthetic AMPs were effective in amelioration of the pulmonary inflammatory response in mice subjected to intra-tracheal MRSA. No bacterial cultures were performed, however, and the positive effects may have been simply anti-inflammatory and not antibacterial. An engineered AMP prevented biofilm formation on human bronchial epithelial cells and protected mice from invasive infection after intra-tracheal Pseudomonas aeruginosa instillation [77]. Pseudomonas aeruginosa continues to be a major resistant pathogen in ventilatory-associated pulmonary infection, and studies have consistently demonstrated activity of AMPs against these organisms [78,79]. Given the previous experience with daptomycin, pharmacokinetic profiles for the treatment of pneumonia with AMPs will need to be given special attention.

Clostridium difficile disease

One of the great mysteries of the human being is the balanced relation that exists for most individuals between the enormous population of gut microflora and the intestinal barrier. Populations of as much as 10¹² organisms/mL of surface mucus coexist with the mucosal endothelium in a symbiotic relation [80]. Antimicrobial peptides from gut epithelial cells are considered major contributors to maintenance of the necessary gut colonization without loss of microbial domain into host tissues [81 –83]. It is a delicate relation among the variables of nutrient intake, ingested foreign microbes, and host regulation of the gut microbiome to avoid the diseases of dysbiosis [84].

For surgeons, the major disease of dysbiosis of the gastrointestinal tract in the 21^st Century is Clostridium difficile infection. Ingested C. difficile spores have an uneventful transit through the intestinal tract when the microbiome of the human colon is in harmony. Disruption of the symbiotic relation among the numerous microbial species, and between the microbial colonists and the host, leads to the vegetative proliferation of C. difficile derived from spores, adhesion to colonic cells, and the elaboration of enterotoxins.

The cationic peptide nisin from the colonic anaerobe Lactococcus lactis has in vitro activity against C. difficile [85]. Reduced spore viability also has been observed with nisin [86]. A semi-synthetic AMP has demonstrated in vitro activity comparable to or better than that of conventional antibiotic treatments for C. difficile [87]. In vivo studies are needed to evaluate the clinical role of AMPs or AMP-focused probiotic therapy for these infections.

Discussion

A large volume of literature published over the last several decades has extolled the virtues of AMPs as treatment for clinical infections. Although a small number of AMPs have made it to clinical practice, the potential seems far greater than what has been achieved to this point. A number of AMP preparations are in clinical trials at this time, but most are topical applications and would not have the scope of application comparable to systemic conventional antibiotic therapy (Table 2) [88,89]. Antimicrobial peptides have been advocated as markers for the diagnosis of infection [90,91], as immune modulators [92], and even for cancer diagnosis/treatment [93,94]. The possible applications of AMPs are increasing in number much faster than clinical evidence is supporting the use of the peptides.

Table 2.

List of Antimicrobial Peptides Proposed for Clinical Trials [88,89]

AMP	Source	Indication	Formulation
Iturin A	Bacteria	Antifungal	Not determined
Ramoplanin	Bacteria	Clostridium difficile infection	Oral administration
WAP-8294A2	Bacteria	Gram-positive bacteria	Systemic
Pexiganin	African clawed frog	Diabetic foot ulcers	Topical cream
Omiganin	Bovine	Catheter infections	Topical gel
Iseganan	Porcine	Oral mucositis	Oral solution
Pexiganin	African clawed frog	Diabetic foot ulcers	Topical cream
Lytixar (LTX-109)	Synthetic	Gram-positive skin/nasal decontamination	Hydrogel
LL-37	Human	Venous leg ulcers	Topical solution
CZEN-002	Human	Vaginal candidiasis	Gel
hLF1-11	Human	Bacterial/fungal infection, immunocompromised stem cell transplants	Systemic
Norexatin	Human	Onychomycosis	Topical
PAC-113	Human	Oral candidiasis	Mouth rinse
PXL01	Human	Prevention of post-operative adhesions; hand surgery	Hydrogel

The theoretical opportunities and liabilities of AMPs as prevention and treatment strategies for infection are identified in Table 3 and have been referred to in this manuscript [95 –97]. The biggest opportunity resides in the abundance of naturally occurring AMPs that provide the templates for defining the biochemical specificity of targeting specific organisms. Templates of antimicrobial activity can then be enhanced with molecular modeling [98], and the amino acid length of potential AMPs can be shortened with computer simulations to create less expensive and more pharmacodynamically advantageous peptides [99]. The peptide secondary structure can be exploited for the design of semi-synthetic and completely synthetic constructs with targeted focus for specific pathogens. The current evidence indicates that naturally occurring AMPs can be active against strains highly resistant to conventional antibiotics. This activity can be enhanced further when consideration is given to the number of amino acids that are available and the number of different residue numbers that can be incorporated into a final peptide sequence.

Table 3.

Opportunities and Vulnerabilities of Antimicrobial Peptides in Management of Surgical Infections

Opportunity

Do not require metabolically active organisms for antimicrobial effect

Rapid killing kinetics

Low potential for resistance

A large cohort of naturally occurring peptides

Antimicrobial templates with amino acid combinations and secondary structures provide an enormous opportunity for semi-synthetic and completely synthetic peptides

Synergism with conventional antibiotics

Neutralize endotoxin

Immune modulation effects

Vulnerability

Inhibition of activity with acid pH, saline, serum/plasma

Lack of research in clinically relevant animal models

Stability in storage

Pharmacokinetic issues

Toxicity/safety concerns

Inadequate delivery systems

High production costs

The liabilities of AMPs are also noted in Table 3. Greater stability of peptide preparations and better delivery systems will be required [100,101]. Isolation and purification of naturally occurring AMPs has been difficult. The opportunities of AMPs are also a liability, as the cost of synthetic construction of particular AMPs with specific microbial targets will be formidable. The proposed design of AMP therapy has the appearance of being very similar to that of conventional antibiotics, and the short generation time of bacteria means that evolving microbial resistance will be an issue that will require continuous monitoring.

As the development of resistance to conventional antibiotics continues at a rapid pace, the investigation into AMPs needs to evolve into clinical trials to define the role they can serve in future prevention and treatment of infection. The extensive literature on AMPs and the potential activity these peptides have against bacterial pathogens that are resistant to convention antibiotics suggests a major role in surgical patients in the future. Applications of AMPs will require investigation into the specific issues of infection in these patients.

Footnotes

Author Disclosure Statement

The author has no conflicts of interest related to this article.

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