antimicrobial peptides antimicrobial peptides antimicrobial peptides (amps), also called host defence peptides (hdps) are part of the innate immune response found among all classes of life. fundamental differences exist between prokaryotic and eukaryotic cells that may represent targets for antimicrobial peptides. these peptides are potent, broad spectrum antibiotics which demonstrate potential as novel therapeutic agents. antimicrobial peptides have been demonstrated to kill gram negative and gram positive bacteria, enveloped viruses, fungi and even transformed or cancerous cells. unlike the majority of conventional antibiotics it appears that antimicrobial peptides frequently destabilize biological membranes, can form transmembrane channels, and may also have the ability to enhance immunity by functioning as immunomodulators. various structures of antimicrobial peptides structure antimicrobial peptides from animals, plants and fungi organised by their secondary structure content. circle size indicates overall molecular weight of each peptide. antimicrobial peptides are a unique and diverse group of molecules, which are divided into subgroups on the basis of their amino acid composition and structure. antimicrobial peptides are generally between 12 and 50 amino acids. these peptides include two or more positively charged residues provided by arginine, lysine or, in acidic environments, histidine, and a large proportion (generally >50%) of hydrophobic residues. the secondary structures of these molecules follow 4 themes, including i) α-helical, ii) β-stranded due to the presence of 2 or more disulfide bonds, iii) β-hairpin or loop due to the presence of a single disulfide bond and/or cyclization of the peptide chain, and iv) extended. many of these peptides are unstructured in free solution, and fold into their final configuration upon partitioning into biological membranes. it contains hydrophilic amino acid residues aligned along one side and hydrophobic amino acid residues aligned along the opposite side of a helical molecule. this amphipathicity of the antimicrobial peptides allows them to partition into the membrane lipid bilayer. the ability to associate with membranes is a definitive feature of antimicrobial peptides, although membrane permeabilization is not necessary. these peptides have a variety of antimicrobial activities ranging from membrane permeabilization to action on a range of cytoplasmic targets. typecharacteristicamps anionic peptidesrich in glutamic and aspartic acidsmaximin h5 from amphibians, dermcidin from humans linear cationic α-helical peptideslack in cysteinececropins, andropin, moricin, ceratotoxin and melittin from insects, magainin, dermaseptin, bombinin, brevinin-1, esculentins and buforin ii from amphibians, cap18 from rabbits, ll37 from humans cationic peptide enriched for specific amino acidrich in proline, arginine, phenylalanine, glycine, tryptophanabaecin and drosocin, apidaecin, diptericin, and attacin from insects, prophenin from pigs, indolicidin from cattle. anionic/cationic peptides forming disulfide bondscontain 1~3 disulfide bond 1 bond: brevinins; 2 bonds: protegrin from pig, tachyplesins from horseshoe crabs; 3 bonds: defensins from humans; more than 3: drosomycin in fruit flies activities the modes of action by antimicrobial peptides the modes of action by which antimicrobial peptides kill microbes are varied, and may differ for different bacterial species. some antimicrobial peptides kill both bacteria and fungi, e.g., psoriasin kills e. coli and several filamentous fungi. the cytoplasmic membrane is a frequent target, but peptides may also interfere with dna and protein synthesis, protein folding, and cell wall synthesis. the initial contact between the peptide and the target organism is electrostatic, as most bacterial surfaces are anionic, or hydrophobic, such as in the antimicrobial peptide piscidin. their amino acid composition, amphipathicity, cationic charge and size allow them to attach to and insert into membrane bilayers to form pores by ‘barrel-stave’, ‘carpet’ or ‘toroidal-pore’ mechanisms. alternately, they may penetrate into the cell to bind intracellular molecules which are crucial to cell living. intracellular binding models includes inhibition of cell wall synthesis, alteration of the cytoplasmic membrane, activation of autolysin, inhibition of dna, rna, and protein synthesis, and inhibition of certain enzymes. in many cases, the exact mechanism of killing is not known. one emerging technique for the study of such mechanisms is dual polarisation interferometry. in contrast to many conventional antibiotics these peptides appear to be bactericidal instead of bacteriostatic. in general the antimicrobial activity of these peptides is determined by measuring the minimal inhibitory concentration (mic), which is the lowest concentration of drug that inhibits bacterial growth. amps can possess multiple activities including anti-gram-positive bacterial, anti-gram-negative bacterial, anti-fungal, anti-viral, anti-parasitic, and anti cancer activities. a big amp functional analysis indicates that among all amp activities, amphipathicity and charge, two major properties of amps, best distinguish between amps with and without anti-gram-negative bacterial activities. this implies that being amps with anti-gram-negative bacterial activities may prefer or even require strong amphipathicity and net positive charge. immunomodulation in addition to killing bacteria directly they have been demonstrated to have a number of immunomodulatory functions that may be involved in the clearance of infection, including the ability to alter host gene expression, act as chemokines and/or induce chemokine production, inhibiting lipopolysaccharide induced pro-inflammatory cytokine production, promoting wound healing, and modulating the responses of dendritic cells and cells of the adaptive immune response. animal models indicate that host defense peptides are crucial for both prevention and clearance of infection. it appears as though many peptides initially isolated as and termed "antimicrobial peptides" have been shown to have more significant alternative functions in vivo (e.g. hepcidin). dusquetide for example is an immunomodulator that acts through p62, a protein involved in toll like receptor based signalling of infection. the peptide is being examined in a phase iii clinical trial by soligenix (sgnx) to ascertain if it can assist in repair of radiation-induced damage to oral mucosa arising during cancer radiotherapy of the head and neck. mechanisms of action scanning electron microscopic images (50,000x magnification) displaying the action of an experimental antimicrobial peptide (nn2_0050) on the cell membrane of e. coli (k12 mg1655) above: intact cell membranes in the control group. below: disrupted cell membranes and leakage of bacterial chromosome (green) in the treated group. antimicrobial peptides generally have a net positive charge, allowing them to interact with the negatively charged molecules exposed on bacteria and cancer cell surfaces, such as phospholipid phosphatidylserine, o-glycosylated mucins, sialylated gangliosides, and heparin sulfates. the mechanism of action of these peptides varies widely but can be simplified into two categories: membranolytic and non-membranolytic antimicrobial peptides. the disruption of membranes by membranolytic antimicrobial peptides can be described by four models: barrel-stave model: the barrel-stave model proposes that amps interact with the lipid bilayer of the microbial cell membrane to form transmembrane channels or "barrel staves". these channels are thought to disrupt the membrane's integrity, leading to the death of the microbe. carpet model: the carpet model proposes that amps adsorb onto the lipid bilayer of the microbial cell membrane, forming a dense layer that causes the membrane to become permeabilized. this model suggests that the amp acts as a "carpet" that covers the surface of the cell, preventing the microbe from functioning properly. toroidal model: the toroidal model proposes that amps interact with the lipid bilayer of the microbial cell membrane to form toroidal structures, which are thought to pinch off sections of the membrane and lead to the formation of vesicles. this process is thought to disrupt the membrane's integrity and cause the death of the microbe. disordered toroidal-pore model: according to this model, the disordered amps wrap around the lipid bilayer and create a pore, which disrupts the membrane's integrity and leads to the death of the microbe. unlike the toroidal model, which suggests that the amp creates a stable toroidal structure, the disordered toroidal-pore model suggests that the amp is flexible and does not form a stable toroidal structure. the peptide-lipid pore complex becomes intrinsically disordered, with the orientation of the peptide not well defined. schematic representation of the amps mechanisms of action when disrupting membranes. several methods have been used to determine the mechanisms of antimicrobial peptide activity. in particular, solid-state nmr studies have provided an atomic-level resolution explanation of membrane disruption by antimicrobial peptides. in more recent years, x-ray crystallography has been used to delineate in atomic detail how the family of plant defensins rupture membranes by identifying key phospholipids in the cell membranes of the pathogen. human defensins have been thought to act through a similar mechanism, targeting cell membrane lipids as part of their function. in fact human beta-defensin 2 have now been shown to kill the pathogenic fungi candida albicans through interactions with specific phospholipids. from the computational point of view, molecular dynamics simulations can provide detailed information about the structure and dynamics of the peptide-membrane interactions, including the orientation, conformation, and insertion of the peptide in the membrane, as well as specific peptide interactions with lipids, ions and solvent. methodsapplications microscopyto visualize the effects of antimicrobial peptides on microbial cells atomic emission spectroscopyto detect loss of intracellular potassium (an indication that bacterial membrane integrity has been compromised) fluorescent dyesto measure ability of antimicrobial peptides to permeabilize membrane vesicles ion channel formationto assess the formation and stability of an antimicrobial-peptide-induced pore circular dichroism and orientated circular dichroismto measure the orientation and secondary structure of an antimicrobial peptide bound to a lipid bilayer dual polarization interferometryto measure the different mechanisms of antimicrobial peptides solid-state nmr spectroscopyto measure the secondary structure, orientation and penetration of antimicrobial peptides into lipid bilayers in the biologically relevant liquid-crystalline state neutron and x-ray diffractionto measure the diffraction patterns of peptide-induced pores within membranes in oriented multilayers or liquids molecular dynamics simulations to study the molecular behaviour and search for specific peptide-lipid interactions mass spectrometry to measure the proteomic response of microorganisms to antimicrobial peptides therapeutic research and use antimicrobial peptides have been used as therapeutic agents; their use is generally limited to intravenous administration or topical applications due to their short half-lives. as of january 2018 the following antimicrobial peptides were in clinical use: bacitracin for pneumonia, topical boceprevir, hepatitis c (oral, cyclic peptide) dalbavancin, bacterial infections, iv daptomycin, bacterial infections, iv enfuvirtide, hiv, subcutaneous injection oritavancin, bacterial infections, iv teicoplanin, bacterial infections, iv telaprevir, hepatitis c, oral cyclic peptide telavancin, bacterial infection, iv vancomycin, bacterial infection, iv. guavanin 2, bacterial infection against gram-positive and gram-negative also. activity beyond antibacterial functions amps have been observed having functions other than bacterial and fungal killing. these activities include antiviral effects, but also roles in host defence such as anticancer functions and roles in neurology. this has led to a movement for re-branding amps as "host-defence peptides" to encompass the broad scope of activities amps can have. anticancer properties some cecropins (e.g. cecropin a, and cecropin b) have anticancer properties and are called anticancer peptides (acps).: 3 hybrid acps based on cecropin a have been studied for anticancer properties.: 7.1 the fruit fly defensin prevents tumour growth, suspected to bind to tumour cells owing to cell membrane modifications common to most cancer cells, such as phosphatidylserine exposure. antibiofilm properties cecropin a can destroy planktonic and sessile biofilm-forming uropathogenic e. coli (upec) cells, either alone or when combined with the antibiotic nalidixic acid, synergistically clearing infection in vivo (in the insect host galleria mellonella) without off-target cytotoxicity. the multi-target mechanism of action involves outer membrane permeabilization followed by biofilm disruption triggered by the inhibition of efflux pump activity and interactions with extracellular and intracellular nucleic acids. other research recently there has been some research to identify potential antimicrobial peptides from prokaryotes, aquatic organisms such as fish, and shellfish, and monotremes such as echidnas. selectivity in the competition of bacterial cells and host cells with the antimicrobial peptides, antimicrobial peptides will preferentially interact with the bacterial cell to the mammalian cells, which enables them to kill microorganisms without being significantly toxic to mammalian cells. with regard to cancer cells, they themselves also secrete human antimicrobial peptides including defensin, and in some cases, they are reported to be more resistant than the surrounding normal cells. therefore, we cannot conclude that selectivity is always high against cancer cells. factors there are some factors that are closely related to the selectivity property of antimicrobial peptides, among which the cationic property contributes most. since the surface of the bacterial membranes is more negatively charged than mammalian cells, antimicrobial peptides will show different affinities towards the bacterial membranes and mammalian cell membranes. in addition, there are also other factors that will affect the selectivity. it's well known that cholesterol is normally widely distributed in the mammalian cell membranes as a membrane stabilizing agents but absent in bacterial cell membranes; and the presence of these cholesterols will