Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria?

Antimicrobial peptides are an abundant and diverse group of molecules that are produced by many tissues and cell types in a variety of invertebrate, plant and animal species. 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. Although these models are helpful for defining mechanisms of antimicrobial peptide activity, their relevance to how peptides damage and kill microorganisms still need to be clarified. Recently, there has been speculation that transmembrane pore formation is not the only mechanism of microbial killing. In fact several observations suggest that translocated peptides can alter cytoplasmic membrane septum formation, inhibit cell-wall synthesis, inhibit nucleic-acid synthesis, inhibit protein synthesis or inhibit enzymatic activity. In this review the different models of antimicrobial-peptide-induced pore formation and cell killing are presented.     

Antimicrobial peptides: biochemical determinants of activity and biophysical techniques of elucidating their functionality

Antimicrobial peptides (AMPs) have been established over millennia as powerful components of the innate immune system of many organisms. Due to their broad spectrum of activity and the development of host resistance against them being unlikely, AMPs are strong candidates for controlling drug-resistant pathogenic microbial pathogens. AMPs cause cell death through several independent or cooperative mechanisms involving membrane lysis, non-lytic activity, and/or intracellular mechanisms. Biochemical determinants such as peptide length, primary sequence, charge, secondary structure, hydrophobicity, amphipathicity and host cell membrane composition together influence the biological activities of peptides. A number of biophysical techniques have been used in recent years to study the mechanisms of action of AMPs. This work appraises the molecular parameters that determine the biocidal activity of AMPs and overviews their mechanisms of actions and the diverse biochemical, biophysical and microscopy techniques utilised to elucidate these.     

Role of lipids in the interaction of antimicrobial peptides with membranes

Antimicrobial peptides (AMPs) take part in the immune system by mounting a first line of defense against pathogens. Recurrent structural and functional aspects are observed among peptides from different sources, particularly the net cationicity and amphipathicity. However, the membrane seems to be the key determinant of their action, either as the main target of the peptide action or by forming a barrier that must be crossed by peptides to target core metabolic pathways. More importantly, the specificity exhibited by antimicrobial peptides relies on the different lipid composition between pathogen and host cells, likely contributing to their spectrum of activity. Several mechanisms of action have been reported, which may involve membrane permeabilization through the formation of pores, membrane thinning or micellization in a detergent-like way. AMPs may also target intracellular components, such as DNA, enzymes and even organelles. More recently, these peptides have been shown to produce membrane perturbation by formation of specific lipid-peptide domains, lateral phase segregation of zwitterionic from anionic phospholipids and even the formation of non-lamellar lipid phases. To countermeasure their activity, some pathogens were successful in developing effective mechanisms of resistance to decrease their susceptibility to AMPs. The functional and integral knowledge of such interactions and the clarification of the complex interplay between molecular determinants of peptides, the pathogen versus host cells dichotomy and the specific microenvironment in which all these elements convene will contribute to an understanding of some elusive aspects of their action and to rationally design novel therapeutic agents to overcome the current antibiotic resistance issue.     

Methodology for identification of pore forming antimicrobial peptides from soy protein subunits β-conglycinin and glycinin

Antimicrobial peptides (AMPs) inactivate microbial cells through pore formation in cell membrane. Because of their different mode of action compared to antibiotics, AMPs can be effectively used to combat drug resistant bacteria in human health. AMPs can also be used to replace antibiotics in animal feed and immobilized on food packaging films. In this research, we developed a methodology based on mechanistic evaluation of peptide-lipid bilayer interaction to identify AMPs from soy protein. Production of AMPs from soy protein is an attractive, cost-saving alternative for commercial consideration, because soy protein is an abundant and common protein resource. This methodology is also applicable for identification of AMPs from any protein. Initial screening of peptide segments from soy glycinin (11S) and soy β-conglycinin (7S) subunits was based on their hydrophobicity, hydrophobic moment and net charge. Delicate balance between hydrophilic and hydrophobic interactions is necessary for pore formation. High hydrophobicity decreases the peptide solubility in aqueous phase whereas high hydrophilicity limits binding of the peptide to the bilayer. Out of several candidates chosen from the initial screening, two peptides satisfied the criteria for antimicrobial activity, viz. (i) lipid-peptide binding in surface state and (ii) pore formation in transmembrane state of the aggregate. This method of identification of antimicrobial activity via molecular dynamics simulation was shown to be robust in that it is insensitive to the number of peptides employed in the simulation, initial peptide structure and force field. Their antimicrobial activity against Listeria monocytogenes and Escherichia coli was further confirmed by spot-on-lawn test.     

Antimicrobial peptides: new candidates in the fight against bacterial infections

Antimicrobial peptides (AMPs) of innate origin are agents of the most ancient form of defense systems. They can be found in a wide variety of species ranging from bacteria through insects to humans. Through the course of evolution, host organisms developed arsenals of AMPs that protect them against a large variety of invading pathogens including both Gram-negative and Gram-positive bacteria. At a time of increasing bacterial resistance, AMPs have been the focus of investigation in a number of laboratories worldwide. Although recent studies show that some of the peptides are likely to have intracellular targets, the vast majority of AMPs appear to act by permeabilization of the bacterial cell membrane. Their activity and selectivity are governed by the physicochemical parameters of the peptide chains as well as the properties of the membrane system itself. In this review, we will summarize some of the recent developments that provide us with a better understanding of the mode of action of this unique family of antibacterial agents. Particular attention will be given to the determinants of AMP-lipid bilayer interactions as well as to the different pore formation mechanisms. The emphasis will be on linear AMPs but representatives of cysteine-bridged AMPs will also be discussed.     

Beyond electrostatics: Antimicrobial peptide selectivity and the influence of cholesterol-mediated fluidity and lipid chain length on protegrin-1 activity

Antimicrobial peptides (AMPs) are a promising class of innate host defense molecules for next-generation antibiotics, as they uniquely target and permeabilize membranes of pathogens. This selectivity has been explained by the electrostatic attraction between these predominantly cationic peptides and the bacterial membrane, which is heavily populated with anionic lipids. However, AMP-resistant bacteria have non-electrostatic countermeasures that modulate membrane rigidity and thickness. We explore how variations in physical properties affect the membrane affinity and disruption process of protegrin-1 (PG-1) in phosphatidylcholine (PC) membranes with altered lipid packing densities and thicknesses. From isothermal titration calorimetry and atomic force microscopy, our results showed that PG-1 could no longer insert into membranes of increasing cholesterol amounts nor into monounsaturated PC membranes of increasing thicknesses with similar fluidities. Prevention of PG-1’s incorporation consequently made the membranes more resistant to peptide-induced structural transformations like pore formation. Our study provides evidence that AMP affinity and activity are strongly correlated with the fluidity and thickness of the membrane. A basic understanding of how physical mechanisms can regulate cell selectivity and resistance towards AMPs will aid in the development of new antimicrobial agents.     

Antimicrobial peptides (AMPs): A promising class of antimicrobial compounds

Antimicrobial peptides (AMPs) are compounds, which have inhibitory activity against microorganisms. In the last decades, AMPs have become powerful alternative agents that have met the need for novel anti-infectives to overcome increasing antibiotic resistance problems. Moreover, recent epidemics and pandemics are increasing the popularity of AMPs, due to the urgent necessity for effective antimicrobial agents in combating the new emergence of microbial diseases. AMPs inhibit a wide range of microorganisms through diverse and special mechanisms by targeting mainly cell membranes or specific intracellular components. In addition to extraction from natural sources, AMPs are produced in various hosts using recombinant methods. More recently, the synthetic analogues of AMPs, designed with some modifications, are predicted to overcome the limitations of stability, toxicity and activity associated with natural AMPs. AMPs have potential applications as antimicrobial agents in food, agriculture, environment, animal husbandry and pharmaceutical industries. In this review, we have provided an overview of the structure, classification and mechanism of action of AMPs, as well as discussed opportunities for their current and potential applications.     

Antimicrobial Protegrin-1 Forms Ion Channels: Molecular Dynamic Simulation, Atomic Force Microscopy, and Electrical Conductance Studies

Antimicrobial peptides (AMPs) are an emerging class of antibiotics for controlling health effects of antibiotic-resistant microbial strains. Protegrin-1 (PG-1) is a model antibiotic among β-sheet AMPs. Antibiotic activity of AMPs involves cell membrane damage, yet their membrane interactions, their 3D membrane-associated structures and the mechanism underlying their ability to disrupt cell membrane are poorly understood. Using complementary approaches, including molecular dynamics simulations, atomic force microscopy (AFM) imaging, and planar lipid bilayer reconstitution, we provide computational and experimental evidence that PG-1, a β-hairpin peptide, forms ion channels. Simulations indicate that PG-1 forms channel-like structures with loosely attached subunits when reconstituted in anionic lipid bilayers. AFM images show the presence of channel-like structures when PG-1 is reconstituted in dioleoylphosphatidylserine/palmitoyloleoyl phosphatidylethanolamine bilayers or added to preformed bilayers. Planar lipid bilayer electrical recordings show multiple single channel conductances that are consistent with the heterogeneous oligomeric channel structures seen in AFM images. PG-1 channel formation seems to be lipid-dependent: PG-1 does not easily show ion channel electrical activity in phosphatidylcholine membranes, but readily shows channel activity in membranes rich in phosphatidylethanolamine or phosphatidylserine. The combined results support a model wherein the β-hairpin PG-1 peptide acts as an antibiotic by altering cell ionic homeostasis through ion channel formation in cell membranes.     

Computational resources and tools for antimicrobial peptides

Antimicrobial peptides (AMPs), as evolutionarily conserved components of innate immune system, protect against pathogens including bacteria, fungi, viruses, and parasites. In general, AMPs are relatively small peptides (<10 kDa) with cationic nature and amphipathic structure and have modes of action different from traditional antibiotics. Up to now, there are more than 19 000 AMPs that have been reported, including those isolated from nature sources or by synthesis. They have been considered to be promising substitutes of conventional antibiotics in the quest to address the increasing occurrence of antibiotic resistance. However, most AMPs have modest direct antimicrobial activity, and their mechanisms of action, as well as their structure–activity relationships, are still poorly understood. Computational strategies are invaluable assets to provide insight into the activity of AMPs and thus exploit their potential as a new generation of antimicrobials. This article reviews the advances of AMP databases and computational tools for the prediction and design of new active AMPs. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Antimicrobial properties and death-inducing mechanisms of saccharomycin,a biocide secreted by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

We recently found that Saccharomyces cerevisiae (strain CCMI 885) secretes antimicrobial peptides (AMPs) derived from the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that are active against various wine-related yeast and bacteria. Here, we show that several other S. cerevisiae strains also secrete natural biocide fractions during alcoholic fermentation, although at different levels, which correlates with the antagonistic effect exerted against non-Saccharomyces yeasts. We, therefore, term this biocide saccharomycin. The native AMPs were purified by gel-filtration chromatography and its antimicrobial activity was compared to that exhibited by chemically synthesized analogues (AMP1 and AMP2/3). Results show that the antimicrobial activity of the native AMPs is significantly higher than that of the synthetic analogues (AMP1 and AMP2/3), but a conjugated action of the two synthetic peptides is observed. Moreover, while the natural AMPs are active at pH 3.5, the synthetic peptides are not, since they are anionic and cannot dissolve at this acidic pH. These findings suggest that the molecular structure of the native biocide probably involves the formation of aggregates of several peptides that render them soluble under acidic conditions. The death mechanisms induced by the AMPs were also evaluated by means of epifluorescence microscopy-based methods. Sensitive yeast cells treated with the synthetic AMPs show cell membrane disruption, apoptotic molecular markers, and internalization of the AMPs. In conclusion, our work shows that saccharomycin is a natural biocide secreted by S. cerevisiae whose activity depends on the conjugated action of GAPDH-derived peptides. This study also reveals that S. cerevisiae secretes GAPDH-derived peptides as a strategy to combat other microbial species during alcoholic fermentations.     

Alpha-helical cationic antimicrobial peptides: relationships of structure and function

Antimicrobial peptides (AMPs), with their extraordinary properties, such as broad-spectrum activity, rapid action and difficult development of resistance, have become promising molecules as new antibiotics. Despite their various mechanisms of action, the interaction of AMPs with the bacterial cell membrane is the key step for their mode of action. Moreover, it is generally accepted that the membrane is the primary target of most AMPs, and the interaction between AMPs and eukaryotic cell membranes (causing toxicity to host cells) limits their clinical application. Therefore, researchers are engaged in reforming or de novo designing AMPs as a ‘single-edged sword’ that contains high antimicrobial activity yet low cytotoxicity against eukaryotic cells. To improve the antimicrobial activity of AMPs, the relationship between the structure and function of AMPs has been rigorously pursued. In this review, we focus on the current knowledge of α-helical cationic antimicrobial peptides, one of the most common types of AMPs in nature.     

Experimental and simulation studies reveal mechanism of action of human defensin derivatives

Antimicrobial peptides (AMPs) are naturally occurring promising candidates which can be used as antibiotics against a wide variety of bacteria. The key component for using them as a potent antibiotic is that their mechanism of action is less prone to bacterial resistance. However, the molecular details of their mechanism of action is not yet fully understood. In this study, we try to shed light on the mode of action of AMPs, possible reason behind it, and their interaction with lipid bilayers through experimental as well as molecular dynamics (MD) simulation studies. The focal of our study was Human beta defensin 3 (hBD-3) which is a naturally occurring AMP. We chose three derivatives of hBD-3, namely CHRG01, KSR, and KLR for the detailed analysis presented in this study. These three peptides are evaluated for their antibacterial potency, secondary structure analysis and mechanism of action. The experimental results reveal that these peptides are active against gram positive as well as gram negative bacteria and kill bacteria by forming membrane pores. The MD simulation results correlate well with the antibacterial activity and shed light into the early membrane insertion dynamics. Moreover, the specific amino acids responsible for membrane disruptions are also identified from the MD simulations. Understanding the molecular level interaction of individual amino acids with the lipid bilayer will greatly help in the design of more efficient antimicrobial peptides.     

Effect of physicochemical properties of peptides from soy protein on their antimicrobial activity

Antimicrobial peptides (AMPs) kill microbial cells through insertion and damage/permeabilization of the cytoplasmic cell membranes and has applications in food safety and antibiotic replacement. Soy protein is an attractive, abundant natural source for commercial production of AMPs. In this research, explicit solvent molecular dynamics (MD) simulation was employed to investigate the effects of (i) number of total and net charges, (ii) hydrophobicity (iii) hydrophobic moment and (iv) helicity of peptides from soy protein on their ability to bind to lipid bilayer and their transmembrane aggregates to form pores. Interaction of possible AMP segments from soy protein with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPC/POPG) bilayers, a mimic of bacterial cell membrane, was investigated. Pore formation was insensitive to helicity and occurred for hydrophobicity threshold in the range of −0.3–0 kcal/mol, hydrophobic moment threshold of 0.3 kcal/mol, net charge threshold of 2. Though low hydrophobicity and high number of charges help in the formation of water channel for transmembrane aggregates, insertion of peptides with these properties requires overcome of energy barrier, as shown by potential of mean force calculations, thereby resulting in low antimicrobial activity. Experimental evaluation of antimicrobial activity of these peptides against Gram positive L. monocytogenes and Gram negative E. coli as obtained by spot-on-lawn assay was consistent with simulation results. These results should help in the development of guidelines for selection of peptides with antimicrobial activity based on their physicochemical properties.     

The effect of the placement and total charge of the basic amino acid clusters on antibacterial organism selectivity and potency

Extensive circular dichroism, isothermal titration calorimetry and induced calcein leakage studies were conducted on a series of antimicrobial peptides (AMPs), with a varying number of Lys residues located at either the C-terminus or the N-terminus to gain insight into their effect on the mechanisms of binding with zwitterionic and anionic membrane model systems. Different CD spectra were observed for these AMPs in the presence of zwitterionic DPC and anionic SDS micelles indicating that they adopt different conformations on binding to the surfaces of zwitterionic and anionic membrane models. Different CD spectra were observed for these AMPs in the presence of zwitterionic POPC and anionic mixed 4:1 POPC/POPG LUVs and SUVs, indicating that they adopt very different conformations on interaction with these two types of LUVs and SUVs. In addition, ITC and calcein leakage data indicated that all the AMPs studied interact via very different mechanisms with anionic and zwitterionic LUVs. ITC data suggest these peptides interact primarily with the surface of zwitterionic LUVs while they insert into and form pores in anionic LUVs. CD studies indicated that these compounds adopt different conformations depending on the ratio of POPC to POPG lipids present in the liposome. There are detectable spectroscopic and thermodynamic differences between how each of these AMPs interacts with membranes, that is position and total charge density defines how these AMPs interact with specific membrane models and thus partially explain the resulting diversity of antibacterial activity of these compounds.     

Plant antimicrobial peptides

Plant antimicrobial peptides (AMPs) are a component of barrier defense system of plants. They have been isolated from roots, seeds, flowers, stems, and leaves of a wide variety of species and have activities towards phytopathogens, as well as against bacteria pathogenic to humans. Thus, plant AMPs are considered as promising antibiotic compounds with important biotechnological applications. Plant AMPs are grouped into several families and share general features such as positive charge, the presence of disulfide bonds (which stabilize the structure), and the mechanism of action targeting outer membrane structures.     

Bacteriocins: perspective for the development of novel anticancer drugs

Antimicrobial peptides (AMPs) from prokaryotic source also known as bacteriocins are ribosomally synthesized by bacteria belonging to different eubacterial taxonomic branches. Most of these AMPs are low molecular weight cationic membrane active peptides that disrupt membrane by forming pores in target cell membranes resulting in cell death. While these peptides known to exhibit broad-spectrum antimicrobial activity, including antibacterial and antifungal, they displayed minimal cytotoxicity to the host cells. Their antimicrobial efficacy has been demonstrated in vivo using diverse animal infection models. Therefore, we have discussed some of the promising peptides for their ability towards potential therapeutic applications. Further, some of these bacteriocins have also been reported to exhibit significant biological activity against various types of cancer cells in different experimental studies. In fact, differential cytotoxicity towards cancer cells as compared to normal cells by certain bacteriocins directs for a much focused research to utilize these compounds as novel therapeutic agents. In this review, bacteriocins that demonstrated antitumor activity against diverse cancer cell lines have been discussed emphasizing their biochemical features, selectivity against extra targets and molecular mechanisms of action.

     

Cell-penetrating peptides and antimicrobial peptides: how different are they?

Some cationic peptides, referred to as CPPs (cell-penetrating peptides), have the ability to translocate across biological membranes in a non-disruptive way and to overcome the impermeable nature of the cell membrane. They have been successfully used for drug delivery into mammalian cells; however, there is no consensus about the mechanism of cellular uptake. Both endocytic and non-endocytic pathways are supported by experimental evidence. The observation that some AMPs (antimicrobial peptides) can enter host cells without damaging their cytoplasmic membrane, as well as kill pathogenic agents, has also attracted attention. The capacity to translocate across the cell membrane has been reported for some of these AMPs. Like CPPs, AMPs are short and cationic sequences with a high affinity for membranes. Similarities between CPPs and AMPs prompted us to question if these two classes of peptides really belong to unrelated families. In this Review, a critical comparison of the mechanisms that underlie cellular uptake is undertaken. A reflection and a new perspective about CPPs and AMPs are presented.     

Amphipathic alpha helical antimicrobial peptides.

Antimicrobial peptides (AMPs) that assume an amphipathic alpha helical structure are widespread in nature. Their activity depends on several parameters including the sequence, size, degree of structure formation, cationicity, hydrophobicity and amphipathicity. The analysis of numerous natural AMPs provided representative values for these parameters and led to a sequence template with which to generate potent artificial lead AMPs. Sequences were then varied in a rational manner, using both natural and nonproteinogenic amino acids, to probe the individual roles of each parameter in modulating biological activity. A high cationicity combined with a stabilized amphipathic alpha helical structure conferred enhanced cidal activity towards all the cell types considered, and was a requirement for Gram-positive bacteria and fungi. An elevated helicity also correlated with increased hemolytic activity. The structural requirements for activity against several Gram-negative bacteria were instead considerably less stringent, so that it persisted in peptides in which formation of a helical structure and/or amphipathicity were impeded. Either a reduced charge or a reduced hydrophobicity resulted in generally inactive peptides. These observations, combined with the kinetics of bacterial membrane permeabilization and time-killing are discussed in terms of currently accepted models of action for this type of peptide. The simple guidelines obtained in this study allowed the design of highly active shortened AMPs and may be generally useful in the development of this type of peptides as anti-infective agents.     

Membrane-active peptides and the clustering of anionic lipids

There is some overlap in the biological activities of cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs). We compared nine AMPs, seven CPPs, and a fusion peptide with regard to their ability to cluster anionic lipids in a mixture mimicking the cytoplasmic membrane of Gram-negative bacteria, as measured by differential scanning calorimetry. We also studied their bacteriostatic effect on several bacterial strains, and examined their conformational changes upon membrane binding using circular dichroism. A remarkable correlation was found between the net positive charge of the peptides and their capacity to induce anionic lipid clustering, which was independent of their secondary structure. Among the peptides studied, six AMPs and four CPPs were found to have strong anionic lipid clustering activity. These peptides also had bacteriostatic activity against several strains (particularly Gram-negative Escherichia coli) that are sensitive to lipid clustering agents. AMPs and CPPs that did not cluster anionic lipids were not toxic to E. coli. As shown previously for several types of AMPs, anionic lipid clustering likely contributes to the mechanism of antibacterial action of highly cationic CPPs. The same mechanism could explain the escape of CPPs from intracellular endosomes that are enriched with anionic lipids.